Phase transition biopolymers and methods of use

ABSTRACT

The present disclosure describes environmentally responsive polypeptides capable of displaying stimuli-triggered conformational changes in a reversible or irreversible manner that may be accompanied by aggregation. Polypeptides include a number of repeated motifs and may be elastomeric or non-elastomeric. The polypeptides may be used to deliver therapeutics to a biological site and to develop bioactive polypeptides that are environmentally responsive.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Provisional PatentApplication No. 61/386,002 filed Sep. 24, 2010, the content of which isincorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant no.5R01-GM61232-08 awarded by the National Institute of Health. Thegovernment has certain rights in this disclosure.

BACKGROUND

Elastomeric proteins, and in particular elastin, have been the subjectof extensive investigations aimed at a molecular understanding of thestructure-function relationship among these proteins, their mechanicalproperties, environmental sensitivity and self-assembly properties.These studies indicated a role for disordered protein structures inproteins and protein domains, and the identification of short recurrentpeptides which were capable of forming protein-polymers with similarstructural and environmental properties. A general elastic,environmentally responsive motif Val-Pro-Gly-X-Gly was found in elastin,where X is any amino acid except Proline, and was used to developelastin-like polypeptides (ELPs) for biotechnological and biomedicaluses. Similarly, resilin-like polypeptides displaying mechanicalproperties comparable to the native resilin protein were made.

Silks, on the other hand, constitute a complex family of proteins thatencompasses both elastomeric and non-elastomeric proteins. Elastomericsilks include a highly repetitive GPGGX motif. Studies have indicatedthat there is an absence of Proline residues in non-elastomeric silks,and that β-sheet structures increase in proportion to the GPGGX content.However, the structures adopted by the abundant GPGGX (SEQ ID NO:1)/GPGQQ(SEQ ID NO: 2) and GGX repeat units remain unclear. Littleprogress has been made on the design of recombinant silk-likebiomaterials. In addition, elastin remains the only elastomericrepetitive protein successfully reduced to a short motif capable ofdisplaying both elasticity and environmental sensitivity.

After more than three decades of research since the discovery of theenvironmental sensitivity of elastin monomers (tropoelastin), the morethan two decades since the identification of the canonical elastomericmotif VPGXG (SEQ ID NO: 3), and the almost two decades since thegeneralization of this repeat unit into the canonical ELP motif VPGXG(SEQ ID NO: 3), only a handful of elastin-inspired polypeptidesdeparting from the canonical sequence have been uncovered, namely minormodifications of the canonical motif, such as LPGXG (SEQ ID NO: 4) andIPGXG (SEQ ID NO: 5), and the repeat unit VPAVG (SEQ ID NO: 6). Recentefforts have made use of complex bioinformatics tools to search forsequence conservation, amino acid patterns, and recurrent motifs amongelastin proteins from different species; these studies have hinted atthe potential functional role of the PG dipeptide in elastin (see, e.g,He, D. et al. (2007) Matrix Biology 26:524-540). In a more generalapproach, studies of similarities in Proline and Glycine content betweena large panel of elastomeric proteins from different species includingelastin, resilin, gluten, and silks failed to identify first principlesfor the design of general elastomeric motifs, or to identify and reduceto practice novel motifs responsible for the elasticity and/orenvironmental sensitivity of these proteins. (see, e.g., Rauscher, S. etal. (2006) Structure 14:1667-1676).

SUMMARY

One aspect of the present disclosure describes environmentallyresponsive polypeptides containing at least ten repeats of an amino acidsequence Z₁Z₂PXGZ₃, wherein P is Proline, G is Glycine, X is from 1 to 4amino acids that are not Proline or Glycine, and Z₁, Z₂ and, Z₃ are eachan amino acid is described. Upon stimulation the polypeptide undergoes aconformational change that is accompanied by aggregation.

Another aspect describes environmentally responsive polypeptides thatcontain at least ten repeats of an amino acid sequence Z₁PXGZ₂RGZ₃,wherein P is proline, G is glycine, R is arginine, D is aspartate, X isfrom 0 to 4 amino acids that are not proline or glycine, and Z₁ and Z₂are each an amino acid, and Z₃ is an amino acid such as aspartate (D).Upon stimulation the polypeptide undergoes a conformational change thatis accompanied by aggregation.

Another aspect describes environmentally responsive polypeptidescomprising at least ten sequences selected from VGAPVG, LGAPVG,VPSALYGVG, VGPAVG, VTPAVG, VPSDDYGQG, VPSDDYGVG, TPVAVG, VPSTDYGVG,VPAGVG, VPTGVG, VPAGLG, VPHVG, VHPGVG, VPGAVG, VPGVAG, VRPVG, GRGDSPY,GRGDSPH, GRGDSPV, GRGDSPYG, RPLGYDS, RPAGYDS, GRGDSYP, GRGDSPYQ,GRGNSPYG, GRGDAPYQ, VPHSRNGG, VPHSRNGL, VPGHSHRDFQPVLHLVALNSPLSGGMRG,HTHQDFQPVLHLVALNTPLSGGMRGIRPGG, FEWTPGWYQPYG or a combination thereof.Upon stimulation the polypeptide undergoes a conformational change thatis accompanied by aggregation.

Another aspect describes environmentally responsive polypeptides thatcontain at least ten PG motifs, and at least nine spacer sequencesbetween the PG motifs. The spacer sequences do not include a PG motifand are between five and thirty amino acid residues in length. Uponstimulation the polypeptide undergoes a conformational change that isaccompanied by aggregation.

Another aspect describes an environmentally responsive polypeptide whichupon stimulation undergoes a conformational change that is accompaniedby aggregation and includes at least ten repeats of an amino acidsequence Z₁PXGZ₂Z₃Z₄Z₅, wherein P is proline, G is glycine, X is from 0to 4 amino acids that are not proline or glycine, and Z₁, Z₂, Z₃, Z₄ andZ₅ are each an amino acid. Z₂ may be optionally absent. The amino acidsequence includes an arginine, as well as either a serine or aspartate,or both serine and aspartate, and at least one hydrophobic residueselected from valine, leucine, isoleucine, histidine, tyrosine,tryptophan and alanine. The asparatate may be optionally substitutedwith glutamate.

Another aspect describes methods of effecting a conformational change ina polypeptide by exposing the polypeptide to a stimulus such that thepolypeptide undergoes a conformational change in response to thestimulus. The conformational change may be accompanied by aggregation orsolubilization.

Another aspect describes nanoparticles formed from an environmentallyresponsive polypeptide which may encapsulate a therapeutic for deliveryto a biological site.

Another aspect describes methods of delivering therapeutics tobiological sites by contacting the biological site with a nanoparticleformed from an environmentally responsive polypeptide, wherein thenanoparticle receives a stimulus at the biological site anddisassembles.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component is labeled in every drawing.The foregoing objects, features and advantages of the present disclosurewill become more apparent from a reading of the following description inconnection with the accompanying drawings in which:

FIG. 1 depicts graphs showing the distribution of the VPGXG motif alongthe sequence of elastins from different species: Homo sapiens, Bovine,Zebrafish, Mus musculus and Rattus (FIGS. 1A and B); motifs such asVPGXG (SEQ ID NO: 3), LPGXG (SEQ ID NO: 4) and IPGXG (SEQ ID NO: 5)altogether account for only 10-20% of the amino acids in thesesequences; sequence conservation among species is limited. Mapping ofminima functional P(X_(n))G motifs along the sequences of Bovine andHomo sapiens elastin shows predominance of highly conserved PG motifs(i.e., sharp peaks) covering the entire sequence as evidenced byabundant overlapping peaks. FIG. 1C shows the localization of the VPGXG(SEQ ID NO: 3) motifs as a digital signal where all residues formingpart of the motif have a value of 1 and 0 otherwise; the P(X_(n))Gmotifs are digitized such that the signal is 1 for Proline andsubsequent residues within a continuous P(X_(n))G motif and 0 for Gresidues within the motif or any non-motif amino acid along thesequence.

FIG. 2 depicts graphs showing the mapping of minima functional P(X_(n))Gmotifs and corresponding reversed G(X_(n))P motifs along the sequence(only shown for residues 0-250) of Flagelliform silk (A), elastin (B),gluten (C) and resilin (D). Similar analyses were performed for otherproteins listed in Table 2. The directionality of the motif is evidencedfor all proteins, except Flagelliform silk (and Dragline silk, not shownhere), where GP and PG motifs (i.e., sharp red and blue peaks,respectively) show the same distribution as a result of the abundantGPGXX (SEQ ID NO: 7) motif, which displays both minima motifs.Increasing frequency of P(X_(n))G motifs with larger n values isobserved when comparing the elastomeric proteins Flagelliform silk,gluten, elastin and resilin (FIG. 2 A-D).

FIG. 3 depicts graphs showing quantitation of minima functional motifsamong different elastomeric and non-elastomeric proteins. Occurrence ofP(X_(n))G motifs before (A) and after (B) normalizing to the totallength of the protein. FIG. 3C shows quantitation of VPG, VPGXG(canonical ELP motif; SEQ ID NO: 3) and PG motifs in elastin fromdifferent species. FIG. 3D shows the percentage of P(X_(n))G motifspreceded by Glycine to yield GP(X_(n))G motifs, as shown for n=0, n=1,n=2 and n≧3 (indicated as PGX_(n)G in the figure). FIG. 3E shows theaverage distance between P(X_(n))G motifs with either n=0 or n=1compared with average distance between a control P(X_(n))G motif.

FIG. 4 is a graph showing the hydropathy index (Hi) distribution forresidues surrounding P(X_(n))G motifs (n=0) in elastin sequences fromHomo sapiens, Bovine, Zebrafish and Mus musculus. This analysisconsiders residues one position before (Pm1, for “Proline minus 1residue”) and 2 positions before (Pm2) a Proline residue in a P(X_(n))Gmotif, as well as residues one position after (Gp1, for “Gly plus 1residue”) and two positions after (Gp2) a Gly residue in a P(X_(n))Gmotif. The Hi was defined as proposed by Kyte and Doolittle (1982). Theidentity of the residues at a given position can be easily read fromthis figure (e.g., Gly=−0.4; Val=4.2; A=1.8, etc.). The box delineatesthe 25 and 95 percentile, and the mean hydropathy is indicated by asquare; raw data is also included for each residue position as filleddiamonds.

FIG. 5 is a graph showing hydropathy index (Hi) distribution forresidues surrounding P(X_(n))G motifs (n=0) in elastin (Homo sapiens),resilin, gluten, collagen (type IIα1, Col2A1), fibronectin, titin,Flagelliform and Dragline silks. This analysis considers residues oneposition before (Pm1, for “Proline minus 1 residue”) and 2 positionsbefore (Pm2) a Proline residue on a P(X_(n))G motif, as well as residuesone position after (Gp1, for “Gly plus 1 residue”) and two positionsafter (Gp2) a Gly residue in a P(X_(n))G motif. The Hi was defined asproposed by Kyte and Doolittle (1982). The identity of the residues at agiven position can be easily read from this figure (e.g., Gly=−0.4;Val=4.2; A=1.8, etc.). The box delineates the 25 and 95 percentile, andthe mean hydropathy is indicated by a square; raw data is also includedfor each residue position as filled diamonds.

FIG. 6 is a graph showing hydropathy index (Hi) distribution forresidues comprising and surrounding P(X_(n))G motifs (n=1) in collagen.In addition to Pm2, Pm1, Gp1 and Gp2, this analysis considers theresidue Pp1 (for “Pro plus 1 position”), which is equivalent to the Xresidue constituting the P(X_(n))G motif. The Hi was defined as proposedby Kyte and Doolittle (1982). The identity of the residues at a givenposition can be easily read from this figure (e.g., Gly=−0.4; Val=4.2;A=1.8, etc.). The box delineates the 25 and 95 percentile, and the meanhydropathy is indicated by a square, raw data is also included for eachresidue position as filled diamonds.

FIG. 7 is a graph showing hydropathy index (Hi) distribution forresidues comprising and surrounding P(X_(n))G motifs (n=4) in resilinand titin. Whereas resilin shows a very tight Hi distribution in allpositions comprising and surrounding the P(X₄)G motif, accounting for29.5% of the full protein sequence, the same motif accounts for only1.6% of the amino acid sequence of titin, which was used for comparisondue to its elasticity and remarkable length (26916 residues).Fibronectin and Col2A1 (Homo sapiens) have 0.12% and 0%, respectively.Titin shows a broad distribution of Hi indices, with the exception ofGp1 where only Gly occurs. In addition to Pm2, Pm1, Gp1 and Gp2, thisanalysis considers the residues Pp1 (for “Pro plus 1 position”), Pp2,Pp3, Pp4, which are equivalent to the 4× residues constituting theP(X_(n))G motif. The Hi was defined as proposed by Kyte and Doolittle(1982). The identity of the residues at a given position can be easilyread from this figure (e.g., Glu=−0.4; Val=4.2; A=1.8, etc.). The boxdelineates the 25 and 95 percentile, and the men hydropathy is indicatedby a square; raw data is also included for each residue position asfilled diamonds.

FIG. 8 depicts graphs showing environmental sensitivities of EIPs withZ₁Z₂PGZ₃Z₄ motif, exemplified for both hexapeptide (A) and pentapeptide(B) repeat units. This family of EIPs displays inverse phase transitionbehavior characterized by a sharp phase separation and self-assemblyupon heating above the LCST or Tt of these polypeptides. The number ofrepeats in each protein-polymer is indicated in the legend. All sampleswere prepared at a concentration of 50 μM in PBS, with the exception of(APGVGP) and (TVPGAG) that were diluted in PBS supplemented with 2MNaCl, at 50 and 100 μM, respectively.

FIG. 9 depicts graphs and a photograph showing the characterization of alibrary of EIPs with the AVPGVG (SEQ ID NO: 8) repeat. Turbidityprofiles for 5 constructs in this library at 25 μM in PBS (A) and PBSsupplemented with 1M NaCl (B). The transition temperatures calculatedfrom (B) varied linearly as the reciprocal of molecular weight of theEIP as expected for canonical ELP sequences (C). The distribution ofmolecular weights in this library is illustrated in (D), where EIP-MW1corresponds to the lowest and EIP-MW5 to the highest molecular weight.

FIG. 10 depicts graphs showing reversible phase transition behaviordisplayed by EIPs with Z₁Z₂PGZ₃Z₄ (SEQ ID NO: 9) motif. This family ofEIPs displays reversible inverse phase transition behavior characterizedby a sharp phase separation and self-assembly upon heating above theLCST or Tt of these polypeptides, followed by disassembly upon loweringthe temperature below the LCST (A). Furthermore, upon heating in asecond heating cycle (B), these EIPs retain their environmentalsensitivity. The number of repeats in each protein-polymer is indicatedin the legend. All samples were prepared at a concentration of 50 μM inPBS, with the exception of (TVPGAG; SEQ ID NO: 10) that was diluted to100 μM in PBS supplemented with 2M NaCl.

FIG. 11 depicts graphs showing reversible and heat-irreversible phasetransition behavior displayed by some EIPs with Z₁Z₂PGZ₃Z₄ (SEQ ID NO:9) motif. This family of EIPs displays inverse phase transition behaviorcharacterized by a sharp phase separation and self-assembly upon heatingabove their LCST or Tt, which may be reversible or irreversible fortemperatures below or above a threshold temperature (A-D), and which mayalso be concentration dependent (C). The number of repeats in eachprotein-polymer is shown in the legend. Unless indicated in the legend,the samples were prepared at 50 μM in PBS.

FIG. 12 depicts graphs showing results of environmental sensitivity ofEIPs with Z₁Z₂PGZ₃Z₄ (SEQ ID NO: 9) motif, exemplified for bothhexapeptide (A) and pentapeptide (B) repeat units. This family of EIPsdisplays inverse phase transition behavior characterized by a sharpphase separation and self-assembly upon heating above the LCST or Tt ofeach polypeptide. The number of repeats in each protein-polymer isindicated in the legend. All samples were prepared at a concentration of50 μM in PBS, with the exception of the randomized EIP ([(ZZPXGZ)₅]-₄)[(SEQ ID NO: 11)₅]-₄ with sequence (GAPFGFAIPMGAGFPTGGLAPFGMGLPAGM)₄,(SEQ ID NO: 12)₄ which was prepared in PBS with 6 M Urea and 1M NaCl.

FIG. 13 depicts graphs showing the reversible and heat-irreversiblephase transition behavior displayed by different EIPs with Z₁Z₂PX₁GZ₃Z₄(SEQ ID NO: 13) motif. This family of EIPs displays inverse phasetransition behavior characterized by a sharp phase separation andself-assembly upon heating above the polypeptides LCST or Tt, which maybe reversible (A, B, D, E), or irreversible (C, F) for temperaturesabove a threshold temperature or for buffer ionic strengths above athreshold ionic strength (E). The number of repeats in eachprotein-polymer is shown in the legend. Unless indicated in the legend,the samples were prepared in PBS at 50 μM.

FIG. 14 is a graph showing results of environmental sensitivity of EIPswith Z₁Z₂PX₁X₂GZ₃Z₄ (SEQ ID NO: 14) motif, exemplified for bothhexapeptide and pentapeptide repeat units. This family of EIPs displaysinverse phase transition behavior characterized by a sharp phaseseparation and self-assembly upon heating above the LCST or Tt of eachpolypeptide. The number of repeats in each protein-polymer is indicatedin the legend. All samples were prepared at a concentration of 50 μM inPBS.

FIG. 15 depicts graphs showing the reversible and heat-irreversiblephase transition behavior displayed by different EIPs withZ₁Z₂PX₁X₂GZ₃Z₄ (SEQ ID NO: 14) motif. This family of EIPs displaysinverse phase transition behavior characterized by a sharp phaseseparation and self-assembly upon heating above the polypeptide LCST orTt, which may be reversible (A, B), or irreversible (C, D) fortemperatures above a threshold temperature. The number of repeats ineach protein-polymer is shown in the legend. Unless indicated in thelegend, the samples were prepared in PBS at 50 μM.

FIG. 16 depicts graphs showing reversible phase transition behavior byan EIP with Z₁Z₂PX₁X₂X₃X₄GZ₃Z₄ (SEQ ID NO: 15) motif. This EIP displaysreversible inverse phase transition behavior characterized by a sharpphase separation and self-assembly upon heating above its LCST or Tt,followed by disassembly upon lowering the temperature below the LCST(A-B). Furthermore, upon heating in a second heating cycle (A-B), thisEIP retains its environmental sensitivity. Samples in (A) were preparedat 25 μM in PBS and 8 M Urea, pH 7 (PBSU), and samples in (B) wereprepared in PBS 8 M Urea at pH 9 at the indicated concentrations.

FIG. 17 depicts graphs showing retro-EIPs display thermoresponsivebehavior distinct to the observed in the parent EIP (shown in red)despite having the same hydrophobicity profiles and amino acidside-chain relationships. Occurrence of Gly at Pm1 (GP motif) uponbackbone reversal resulted in a pronounced decrease in the Tt and theemergence of heat-irreversible phase separation upon heating above athreshold temperature (A-B). Noteworthy, the difference in the Ttobserved in (A) is underestimated, as the Tt of (VPGVG)₃₀ is likely tobe ˜10° C. higher than shown, since this construct lacks a His-tagpresent in (VGPVG)₃₀; then, the overall ΔTt in (A) is ˜30° C., which islikely to be similar to the ΔTt observed in (B). Retro-EIP in (C),wherein the parent PX₁G motif was conserved, suggests that overallhydrophobicity is a good predictor of the inverse transition temperatureof Z₁Z₂PX₁GZ₁Z₂ (SEQ ID NO: 13) motifs. The large hysteresis displayedby VPAVG (SEQ ID NO: 6) (See FIG. 15A), is absent in the correspondingretro-EIP (D). Samples were prepared in PBS at 50 μM unless indicated.

FIG. 18 depicts graphs and photographs showing modified retro-EIPsunravel the role of Gly at Pm1 in the environmental sensitivity andself-assembly properties of EIPs comprised of minima functional PX_(n)Gmotifs. Substituting a hydrophobic residue (relative to Gly) for Glyresidue at Pm1, results in an unexpected dramatic increase in the Tt ofthe modified retro-EIP (shown in red), which restores the transitiontemperature to a value closer to that of the parent EIP (FIGS. 17A-B)and the sensitivity to changes in buffer ionic strength (B), andsuppresses heat-irreversible phase transition behavior (A). (C) Phasecontrast microscopic analysis of the structures formed uponself-assembly of EIP constructs drying on glass surfaces. (Ca)Self-assembled fractals formed by EIP (VPAVG)₄₅ (SEQ ID NO: 6)₄₅ with aPX₁X₂G (SEQ ID NO: 16) minima functional motif. (Cb) Retro-EIP(VGPAVG)₂₀ (SEQ ID NO: 17)₂₀ where Pm1 is Gly does not form fractalstructures and assembles into fibrillar-like densely packed structures.(Cc) The modified retro-EIP (VTPAVG)₂₀ (SEQ ID NO: 18)₂₀ shows restoredself-assembly into fractal structures. (Cd) Upon rehydration of theimaged droplets. Large fibrils are observed for the retro-EIP (Cd),while only small submicron aggregates (evidenced in the roughness of thesurface) are observed for the modified retro-EIP (Ce). (Cf)Environmental scanning electron microscopy shows similar fractalstructures formed by an elastin-like polypeptide (with motif VPGXG (SEQID NO: 3), where X=[A:G]) when frozen-dried above its transitiontemperature. Scale bar is 50 μM. Note that threonine is more hydrophobicthan Gly according to Urry's hydrophobicity scale (see, Urry, D. W. etal. (1992) Biopolymers 32:1243-1250).

FIG. 19 is a graph showing self-assembly of EIP with sequence(VTPAVG)₂₀, comprised of a minima functional motif PX₁X₂G (SEQ ID NO:16), into nano-structures. The formation of a third population (P3) (˜4%mass), with hydrodynamic radius larger than 1 mm, for temperatures above38° C., reduced the quality of the DLS data and prevented dataacquisition at higher temperatures. Noteworthy, turbidity profilesdemonstrate stable light scattering properties for temperature between40° C. and 75° C., presumably arising from small micrometer sizedparticles; major aggregation is expected to occur at ˜80° C.,considering that major aggregation of (VPTAVG)₂₅ ((SEQ ID NO: 18)₂₅)occurs at ˜72° C. (FIG. 18B). Sample was prepared at 50 μM in PBS.

FIG. 20 is a graph showing thermoresponsive behavior ofself-cross-linkable in situ gelling materials composed ofelastomeric-inspired polypeptides. Cysteine residues were incorporatedinto EIPs displaying PG and PX₁G motifs to enable theirself-cross-linking via disulfide bonding. The primary sequence of theseprotein polymers is described as (ZVPGXG)₁₄₄ and (VPZGXG)₁₄₄ ((SEQ IDNO: 19)₁₄₄), where [Z,X]=[5A:1C, V], that is 1 Cys every 5 Ala residuesin the Z position, and a Cal in the X position for every repeat, for atotal of 144 repeat units. Noteworthy, the EIP (ZVPGXG)₁₄₄ ((SEQ ID NO:20)₁₄₄) was designed to display the bioactive motif GXXPG (SEQ ID NO:21) responsible for elastin bioactivity. The inverse transitiontemperature of these self-cross-linkable EIPs was engineered to occurbelow body temperature to allow for rapid coacervation before the onsetof gelation via disulfide bonding

FIG. 21. depicts graphs showing multiple Pro and Gly arrangementsbesides the canonical Pro-Gly dipeptide are conducive to unstructuredprotein-polymers that display “smart” behavior. Protein-polymers withperiodic Pro and Gly residues arranged as PX_(n)G units, where n=0 (A),1 (B), 2 (D), 3 and 4 (E), and having pentapeptide, hexapeptide andnonapeptide repeat units display thermally-triggered phase transitionbehavior. These protein-polymers lack ordered secondary structures asshown by their circular dichroism spectra characteristic of disorderedproteins (C). We found that protein-polymers in A, B, D and E fallwithin any of three types of phase transition behavior, corresponding tothree degrees of hysteresis in the reversibility of theirthermally-triggered phase transition: zero, finite and heat-sensitive,infinite hysteresis (F). All turbidity measurements were conducted inPBS at a polypeptide concentration of 50 μM, except for VRPVG (+1MNaCl), VAPGVG (+0.5 M NaCl), APGVG (+2 M NaCl) and VPSALYGVG (+8 Murea). CD studies were conducted in water at a polypeptide concentrationof 5 μM.

FIG. 22. Depicts graphs showing that “smart” biopolymers exhibitprotein-like features. A simple method (A) was applied which wasinspired in the do's and don'ts of non-fibrillar Pro and Gly-richproteins to favor protein disorder, to engineer a protein-sized,240-residue-long protein-polymer with a target hydropathy—equivalent totransition temperature in of 37° C. and composed of 40 hexapeptidemotifs containing Pro-X-Gly units with randomly selected amino acidsspanning a broad range of hydropathies (B). This protein-sizedbiopolymer behaved as an intrinsically disordered protein (C) andexhibited “smart”, phase transition behavior (D). Gly residues precedinga PX_(n)G unit modulated the phase transition behavior of “smart”protein-polymers (E-F). The inset of (F) shows the thermally-triggeredassembly of a n environmentally responsive polypeptide with repeat unitVTPAVG that was not observed for the Gly-variant VGPAVG. (G)Backbone-reversed protein-polymers present identical amino acidpatterns, which were exemplified with the structure of a “smart”pentapeptide motif and its retro-motif as observed in the crystalstructure of two different proteins (PDB id 3MKR_B and 1OZP,respectively). Changes in the phase behavior of “smart” protein-polymerson backbone-reversal (H-I, FIG. S11A) were observed—mostly changes inthe hysteresis of the transition—as well as large changes in theensemble of dynamic conformers that characterize their secondarystructure (K, FIG. S11B-D).

FIG. 23. Depicts graphs showing environmentally responsive polypeptideshaving a syntax that is truly protein-like. First, two protein-polymerswere designed with identical amino acid composition (A), “smart”matrikine 1 (SM1) and SM2, where only SM1 conforms to a bioactive motifGXXPG found in various extracellular matrix proteins. These materialswere engineered to self-gel upon subcutaneous injection by enabling theformation of disulfide bonds upon phase transition through carefullyspaced Cys residues. (B) SM1 and SM2 displayed identical phasetransition behavior in PBS (pH 7.4) and coacervated below bodytemperature. (C) The bioactive SM1 (at 350 μM in PBS) prevented tumorgrowth when used as vehicle for the inoculation of 0.5×10⁵ HT-1080 tumorcells into the leg (n=10) of nude mice, whereas the non-bioactive SM2(at 350 μM in PBS) had no effect on tumor growth. Asterisk (*) indicatesstatistical significance with a 95% confidence using a Bonferroni testto compare the mean tumor volumes after 17 days of tumor inoculation.FIG. 27 shows the anti-tumor activity of SM1-24 for tumors inoculated inthe back of nude mice. In a second example, the complex and long peptidesequences forming the bioactive sites of murine (PDB file: 1DY0) andhuman endostatin (PDB file: 1BNL) (D), which amino acid sequences areshown in blue and red, respectively, are conferred with “smart” behavioron polymerization. These environmentally responsive polypeptides behaveas intrinsically disordered proteins and are highly stable in aqueoussolution (E), and display inverse phase transition behavior (FIG. 38)(F). The ability to design “smart” protein-polymers with monomer unitsthat have defined, local secondary structure propensities, as in humanand murine endostatin (G, within the box), may enable the development ofa broader set of “smart”, drug-like protein-polymers derived from thegrowing list of polypeptide hormones that remain partially disordered onpolymerization (G). Additional peptide hormones of interest werestudied. Secondary structures were predicted using the Jnet algorithm,where ‘H’ is α-helix, ‘E’ is β-sheet, and ‘-’ is random coil. Circulardichroism data were obtained in water at a polypeptide concentration of5 μM. Images of the 3D structures of endostatin were rendered usingPyMOL.

FIG. 24 is a graph showing environmentally responsive “smart” behaviorof a polypeptide with a non-repetitive, 36 amino acid long repeat unit,designed as in FIG. 22A, but using a target hydropathy of 2, accordingto Kyte-Dolittle's scale.

FIG. 25 depicts graphs showing that high Gly content is not aprerequisite for the design of environmentally responsive polypeptides.Gly residues are not a sine qua non element for the design ofprotein-polymers that display fully reversible phase transitionbehavior. Protein-polymer concentration was 50 μM unless otherwiseindicated.

FIG. 26 depicts graphs showing backbone reversal of an environmentallyresponsive polypeptide results in pronounced changes in phase behaviorand secondary structure propensities. (A) Changes in phase behavior fora fourth test-in-case of the effect of backbone reversal on phasebehavior. The retro-polypeptide showed a greater propensity forcoacervation but reduced sensitivity to ionic strength. (C-D) Thecircular dichroism spectra of three pairs of environmentally responsivepolypeptides and their respective retro-motifs revealed significantchanges in the overall disorder of the structures (negative peak at ˜200nm) and β-turn content (negative region around 210 nm). Turbiditystudies were conducted in PBS (with salt concentrations as indicated) ata polypeptide concentration of 50 μM. CD studies were done in water at apolypeptide concentration of 5 μM.

FIG. 27 is a graph showing that a polypeptide containing a matrikinemotif GXXPG, SM1-24 (250 μM in PBS), prevented the grafting of 1×10⁶HT1080 tumor cells inoculated into the back of nude mice. A controlpolypeptide, SM2-24 (250 μM in PBS), with a disrupted motif butidentical phase transition behavior (FIG. 23) had no effect on tumorgrowth. Tumor volumes were measured 19 days after inoculation.

FIG. 28 depicts a graph and photograph showing that environmentallyresponsive polypeptides may be designed to display UCST behavior.

FIG. 29 is a graph showing reversible UCST behavior in PBS whichbehavior may be tuned by polypeptide concentration and the number ofrepeating units.

FIG. 30 is a graph showing bioactive environmentally responsivepolypeptides incorporating the peptide drug GRGDSP.

FIG. 31. is a graph showing the UCST behavior of environmentallyresponsive polypeptides containing RGD tripeptides.

FIG. 32 is a graph showing the UCST behavior of environmentallyresponsive polypeptides may be modulated by electrostatic interactionsbetween positively and negatively charged amino acids within thesequence.

FIG. 33 is a graph showing the UCST behavior of environmentallyresponsive polypeptides does not require electrostatic interactions.

FIG. 34 is a graph showing that RGD-containing environmentallyresponsive polypeptides that display UCST behavior are compatible withmultiple arrangements of Pro and Gly residues.

FIG. 35 is a graph showing that the UCST behavior of environmentallyresponsive polypeptides can be tuned by adjusting the hydrophobicity ofthe residues comprising the repeating unit.

FIG. 36 is a graph showing that environmentally responsive polypeptidesthat contain the peptide drug PHSRN display UCST behavior.

FIG. 37 is a graph showing that environmentally responsive polypeptidesmay be designed to display complex phase behaviors.

FIG. 38 is a graph showing that an environmentally responsivepolypeptide based on the bioactive site of murine Endostatin displayedan inverse phase transition temperature reminiscent of otherenvironmentally responsive polypeptides with simpler syntax. The phasetransition of a 5 μM solution of mEndo1-6 in PBS (pH 6.4) (A) wasaccompanied by a decrease in the disorder of the polypeptideconformation (B), as measured by circular dichroism under identicalconditions as in (A).

DETAILED DESCRIPTION

The present disclosure describes a model for the design of elastomericand non-elastomeric protein-polymers and polypeptides which areenvironmentally responsive, by introducing repeats of functional motifsof the form Z₁Z₂PXGZ₃ (SEQ ID NO: 22) or Z₁Z₂PXGZ₃Z₄ (SEQ ID NO: 22),wherein in each case P is Proline, G is Glycine, X is from 1 to 4 aminoacids that are not Proline or Glycine, and Z₁, Z₂, Z₃ and Z₄ are anyamino acid. In certain embodiments, Z₁, Z₂, Z₃ and Z₄ do not generate aPG (proline-glycine) motif. The present disclosure also describesenvironmentally responsive polypeptides which include ten or moresequences selected from VGAPVG (SEQ ID NO: 24), LGAPVG (SEQ ID NO: 25),VPSALYGVG (SEQ ID NO: 26), VGPAVG (SEQ ID NO: 27), VTPAVG (SEQ ID NO:28), VPSDDYGQG (SEQ ID NO: 29), VPSDDYGVG (SEQ ID NO: 30), TPVAVG (SEQID NO: 31), VPSTDYGVG (SEQ ID NO: 32), VPAGVG (SEQ ID NO: 33), VPTGVG(SEQ ID NO: 34), VPAGLG (SEQ ID NO: 35), VPHVG (SEQ ID NO: 36), VHPGVG(SEQ ID NO: 37), VPGAVG (SEQ ID NO: 38), VPGVAG (SEQ ID NO: 39), VRPVG(SEQ ID NO: 40), GRGDSPY (SEQ ID NO: 41), GRGDSPH (SEQ ID NO: 42),GRGDSPV (SEQ ID NO: 43), GRGDSPYG (SEQ ID NO: 44), RPLGYDS (SEQ ID NO:45), RPAGYDS (SEQ ID NO: 46), GRGDSYP (SEQ ID NO: 47), GRGDSPYQ (SEQ IDNO: 48), GRGNSPYG (SEQ ID NO: 49), GRGDAPYQ (SEQ ID NO: 50), VPHSRNGG(SEQ ID NO: 51), VPHSRNGL (SEQ ID NO: 52), VPGHSHRDFQPVLHLVALNSPLSGGMRG(SEQ ID NO: 53), HTHQDFQPVLHLVALNTPLSGGMRGIRPGG (SEQ ID NO: 54),FEWTPGWYQPYG (SEQ ID NO: 55), or any combination thereof.

Environmentally responsive polypeptides including the motif(Z₁Z₂PGZ₃G)_(n) (SEQ ID NO: 56)_(n) may also be formed, where Z₁, Z₂ andZ₃ are any amino acid. These polypeptides may be bioactive, elastic or acombination thereof. This motif, when repeated consecutively, includesthe bioactive motif GXXPG (SEQ ID NO: 21) responsible for elastin'sability to control various physiological processes includinginflammation, chemotaxis, cell proliferation and differentiation,extracellular matrix remodeling and the like. Polypeptides containingthe motif (Z₁Z₂PGZ₃G)_(n) (SEQ ID NO: 56)_(n) may be elastic andenvironmentally responsive and provide a bioactive signal when used toform recombinant elastin-like materials.

Also described are polypeptides in which the cell adhesion peptideGRGDSP is modified to conform to a P(X_(n))G motif. The phase transitiontemperature of a polypeptide containing such a motif may be controlledby adding or modifying residues incorporated into the motif, such thatthe original cell adhesion signal is modulated to signal, for example,exclusively through integrins or through both integrins and theelastin-binding receptor. Examples of such polypeptides include thosecontaining the octapeptides (GRGDSPZG)_(n) ((SEQ ID NO: 57)_(n)) and(GRGDSPGZ)_(n) ((SEQ ID NO: 58)_(n)), and FEWTPGWYQPY (SEQ ID NO: 59).Environmentally responsive polypeptides may include one or more of a PGmotif, PX₁G motif (where X₁ is an amino acid), or combination thereof.

The protein polymers produced are environmentally responsive, and may beelastomeric or non-elastomeric. The present disclosure demonstratesminima functional motifs that confer environmental responsiveness topolypeptides thereof. The environmental sensitivity may be tuned byvarying polypeptide molecular weight, polypeptide concentration, bufferionic strength, hydrophobicity of amino acids in non-essential positionswithin the motif, the number of residues separating Pro and Gly, and theprecise localization of additional Gly residues which may surround theP(X_(n))G unit. Polypeptides incorporating the motifs may be elastomericor non-elastomeric protein-polymers, and may display reversible inversephase transition behavior and/or heat-irreversible inverse phasetransition above a critical temperature, typically higher than bodytemperature.

Environmentally responsive refers to the property of a given polypeptideto undergo conformational changes, such as coacervation or aggregation,in response to an external stimulus. Aggregation may be reversible orirreversible. The environmentally responsive polypeptide may respond toa small change in stimulus with a pronounced physical change in one ormore properties, such as a sharp change in solubility. Without limitingthe scope of this disclosure, examples of stimuli include changes intemperature, pH, chemicals, electric field, and buffer ionic strength.

Polypeptides described herein may undergo a reversible phase transitionor an irreversible soluble-to-insoluble phase transition in aqueoussolution upon heating through a characteristic transition temperature(lower critical phase transition or LCST). If reversible, the transitiontemperature at which the polypeptide resolubilizes and transitions frominsoluble-to-soluble may be the same as, or different from thesoluble-to-insoluble phase transition temperature. Polypeptides mayexhibit phase separation when exposed to a threshold temperature that isabove a lower critical solution temperature (LCST) of the polypeptide,or may exhibit phase separation when exposed to temperatures below anupper critical solution temperature (UCST) of the polypeptide.Polypeptides described herein may also exhibit phase separation whenexposed to threshold temperatures both above the lower critical solutiontemperature (LCST) and below the upper critical solution temperature(UCST). Phase separation may reversible or irreversible. In certainembodiments, a polypeptide may exhibit a reversible phase separation inresponse to a one stimulus and an irreversible phase separation inresponse to a different stimulus. The different stimulus may bedifferent in type or degree.

The difference between the two transition temperatures may be at leastabout 1° C., at least about 2° C., at least about 3° C., at least about4° C., at least about 5° C., at least about 6° C., at least about 7° C.,at least about 8° C., at least about 9° C., at least about 10° C., atleast about 12° C., or at least about 15° C. The resolubilizationtransition temperature may be higher or lower than thesoluble-to-insoluble phase transition temperature. The polypeptides mayundergo inverse temperature transition, becoming more ordered as thetemperature increases.

The compositions and polypeptides described herein display a surprisingenvironmentally responsive profile. For example, it has been allegedthat the pentapeptide motif VPXVG displays inverse temperaturetransition only if X=A, where A is Alanine (see, e.g., Bessa, P. C. etal. (2010; J Control Release. 142(3):312-8). The inventors surprisinglyfound that this was not the case. The present disclosure introducesfunctional motifs that facilitate formation of stable, ordered,secondary structures in a given dynamic range of stimuli sensed by thepolypeptides (e.g., below the LCST of a polypeptide, or the UCST of apolypeptide). Furthermore, the present disclosure describes elastomericand/or environmentally responsive polypeptides which are proteinpolymers containing pentapeptide, hexapeptide, septaheptide,octapeptide, nonapeptide motifs, or a combination thereof. The motifsmay be at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, at least 27, at least 28, at least 29, at least 30, at least 35, atleast 40, at least 45, at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 100, at least 110, at least 120, at least 130, atleast 140, or at least 150 amino acids in length. Suitable amino acidsinclude naturally occurring amino acids, D-amino acids, L-amino acids,and synthesized amino acids, such as unnatural amino acids. Table 1displays the transition behavior of certain exemplary motifs disclosedherein, with respect to displaying UCST, LCST or both.

TABLE 1 LCST and USCT Behavior of Exemplary Polypeptide MotifsSequence Motifs Transition Behavior VAPVG (SEQ ID NO: 27) LCSTVGAPVG (SEQ ID NO: 24) LCST LGAPVG (SEQ ID NO: 25)VPSALYGVG (SEQ ID NO: 26) LCST VGPAVG (SEQ ID NO: 17) LCSTVTPAVG (SEQ ID NO: 18) LCST TVPGAG (SEQ ID NO: 71) LCSTAVPGVG (SEQ ID NO: 8) TVPGVG (SEQ ID NO: 70)AVPGVGAVPGVGAVPGVGAVPGVGAVPGVGCVPG VG (SEQ ID NO: 64)GAPFGFAIPMGAGFPTGGLAPFGMGLPAGM LCST (SEQ ID NO: 12)VPSDDYGQG (SEQ ID NO: 29) LCST and UCST VPSDDYGVG (SEQ ID NO: 30) LCSTTPVAVG (SEQ ID NO: 31) LCST VPSTDYGVG (SEQ ID NO: 32) LCSTVPAGVG (SEQ ID NO: 33) LCST (for each) VPTGVG (SEQ ID NO: 34)VPAGLG (SEQ ID NO: 35) VPAGVGVPAGVGVPAGVGVPAGVGVPAGVGVPCGVG (SEQ ID NO: 65) GVPAGHRYPIGGGQPHGKGCPDGVFRPVGLGAPYGHGAPNGMHRPLGIGKPRGHMYPKGQGQPMGHLVP DGVGFPRGRKKPVGVGKPIGNGHPIGARTPLGYGMPDGVGMPMGLFLPNGHGAPHGQGYPAGKLIPKGKG HPFGKGRPLGAGRPTGFKMPKGLGKPMGVGQPQGHFVPFGLGQPTGQGAPRGGSQPAGLGHPLGAGAPA GRCHPYGMGVPRGLAMPRGHGQPRGVGYPKGH(positions 5-244 of SEQ ID NO: 105) GVGPAGHRYPIGGQGPHGKCGPDGVFRPVGLAGPYGHAGPNGMHRPLGIKGPRGHMYPKGQQGPMGHLV PDGVFGPRGRKKPVGVKGPIGNHGPIGARTPLGYMGPDGVMGPMGLFLPNGHAGPHGQYGPAGKLIPKGK HGPFGKRGPLGARGPTGFKMPKGLKGPMGVQGPQGHFVPFGLQGPTGQAGPRGGSQPAGLHGPLGAAGP AGRCHPYGMVGPRGLAMPRGHQGPRGVYGPKGH(SEQ ID NO: 110) VPHVG (SEQ ID NO: 36) LCST VHPGVG (SEQ ID NO: 37) LCSTVPGAVG (SEQ ID NO: 38) LCST VPGVAG (SEQ ID NO: 39) LCSTAPGVG (SEQ ID NO: 99) LCST VPGVA (SEQ ID NO: 111) LCSTVRPVG (SEQ ID NO: 40) LCST GRGDSPY (SEQ ID NO: 41) UCST (for each)GRGDSPH (SEQ ID NO: 42) GRGDSPV (SEQ ID NO: 43) GRGDSPYG (SEQ ID NO: 44)UCST RPLGYDS (SEQ ID NO: 45) UCST and LCST (for each)RPAGYDS (SEQ ID NO: 46) GRGDSYP (SEQ ID NO: 47) UCSTGRGDSPYQ (SEQ ID NO: 48) UCST GRGNSPYG (SEQ ID NO: 27) UCSTGRGDAPYQ (SEQ ID NO: 49) UCST (predicted) VPHSRNGG (SEQ ID NO: 51) UCSTVPHSRNGL (SEQ ID NO: 52) VPGHSHRDFQPVLHLVALNSPLSGGMRG LCST(SEQ ID NO: 53) HTHQDFQPVLHLVALNTPLSGGMRGIRPGG LCST (SEQ ID NO: 54)

The polypeptides may include a combination of LCST and UCST motifs whichmay convey both UCST and LCST transition behavior to the polypeptide.The LCST and UCST motifs may be interspersed in the polypeptide. Forexample, the LCST and USCT motifs may be randomly distributed in thepolypeptide, may alternate with each other, may be consecutive, or mayinclude spacer sequences between them, or any possible combinationthereof.

The polypeptides may include at least about 5, at least about 10, atleast about 11, at least about 12, at least about 13, at least about 14,at least about 15, at least about 16, at least about 17, at least about18, at least about 19, at least about 20, or at least about 25 of themotifs described herein and less than about 1000, less than about 500,less than about 400, less than about 300, less than about 275, less thanabout 250, less than about 225, less than about 200, less than about150, less than about 100, less than about 75, less than about 50, orless than about 30 of the motifs described herein. The polypeptides mayinclude only one repeated motif, or may include a number of differentmotifs, as set forth above, which may or may not be repeated.Polypeptides may be formed which are homopolymers of the repeatingunits, or heteropolymers, including alternating copolymers, periodiccopolymers, block copolymers, and statistical copolymers. Block polymersmay include diblocks or triblocks of the motifs described herein.

The motifs may be consecutive within the polypeptide, may be separatedby one or more spacer sequences, or a combination thereof. The spacersequences may include at least 1, at least 2, least 3, at least 4, least5, at least 6, least 7, at least 8, least 9, at least 10, least 11, atleast 12, least 13, at least 14, least 15, at least 16, least 17, atleast 18, least 19, at least 20, least 21, at least 22, least 23, atleast 24, least 25, at least 26, at least 27, at least 28, at least 29,at least 30, at least 35, at least 40, at least 45, at least 50, atleast 60, at least 70, at least 80, at least 90, at least 100, at least150, at least 200, at least 250, or at least 300 amino acids.

Polypeptides may also be retro-polypeptides, including the polypeptidesor one or more of the motifs disclosed herein as a reverse sequencereading from the C-terminus to the N-terminus, rather than theN-terminus to the C-terminus.

The present disclosure facilitates the design of biologically inspiredpolypeptides based on the identification and reverse engineering ofminima functional motifs found in natural repetitive proteins, whichconfer a variety of structural or functional properties. The presentinventors discovered that amino acid sequences seen to recur in naturalrepetitive proteins occur as a subspace of particular sequences, whichhave been selected through evolution, within a larger space of sequencesdisplaying similar properties.

The compositions disclosed herein can be used as materials for a varietyof biomedical and biotechnological applications, including, withoutlimitation, those pertaining to the uses of elastin-derivedbiomaterials, silk-like biomaterials, resilin-like biomaterials andelastin-like biomaterials (see, e.g., U.S. Pat. No. 7,429,458; U.S. Pat.No. 6,852,834; U.S. Pat. No. 5,336,256; U.S. Patent Application No.2010/0015070; U.S. Pat. No. 7,674,882; and U.S. Pat. No. 4,976,734, thedisclosures of each of which are herein incorporated by reference intheir entireties).

Amino acid motifs and polypeptides disclosed herein may be used in thedesign and synthesis of elastomeric-inspired polypeptides, which may beengineered to confer or possess environmental sensitivity and/orelasticity to protein-polymers, while maintaining sequence diversity. Avariety of useful properties and applications are envisaged, includingthose presented below.

The functional motifs described are sufficiently flexible to beincorporated directly into bioactive polypeptides to conferenvironmental sensitivity and elasticity to such polypeptides. Forexample, the present disclosure makes it possible to transform a proteinbioactive peptide signal (e.g., cell adhesion peptides, integrininhibitors, anti-inflammation peptides, cell differentiation,proliferation, angiogenic and anti-angiogentic signals, etc.) into anenvironmentally responsive polypeptide capable of acting as a “smart”drug that is environmentally responsive.

Functional motifs may also be included in combination with one or moreother drugs or therapeutics transported as part of an engineeredself-assembled therapeutic delivery vehicle wherein the vehicle itselfmay constitute an environmentally responsive polypeptide based biodrug.The polypeptide may become bioactive after undergoing a conformationalchange at the biological target site. Functional motifs may also beincluded or introduced into a scaffolding material wherein thefunctional motifs carry biochemical cues for controlling cell-cell andcell-surface interactions, inflammation, chemotaxis or any otherrelevant biological process.

Environmentally responsive, bioactive polypeptides, may be used asintegrin inhibitors for anti-angiogenic therapy or as activepro-angiogenic materials for tissue engineering applications. Thesequence diversity of the polypeptides described herein may be exploitedbecause different integrins recognize alternative peptide sequences. Forexample, α4β1 can recognize EILDV (SEQ ID NO: 60) and REDV (SEQ ID NO:61). Polypeptides described herein having an unordered structure aresuitable for displaying these signals. Anti-inflammatory environmentallyresponsive polypeptides may include one or more of a PG motif, PX₁Gmotif, or combination thereof. The motif may be an anti-inflammatorypeptide, such as FEWTPGWYQPY (SEQ ID NO: 59) and modifications thereof,that prevent scaffold immunorejection.

The polypeptides described herein display similar elasticity andenvironmental sensitivity to other elastin-like polypeptides (ELPs), andmay be utilized in applications which exploit the properties of ELPs.The use of the motifs described herein allows for control of thehydrophobicity profile of the polypeptides or blocks withinblock-copolymer polypeptides, by substituting a variety of residues inany of the positions available for substitution within the functionalmotif. Polypeptides may thus be designed, for example, to self-assembleinto more complex and/or defined structures, to gain control on theelastic force (correlated with Pro content and hydrophobicity), or toimprove the environmental sensitivity of tags for protein purification.

Polypeptides described herein may also be used in drug and therapeuticdelivery applications for treating patients suffering from a disease orcondition. Such vehicle delivery applications may include the formationof nanoparticles from environmentally responsive polypeptides and theirdelivery to a biological site such as an organ, tissue, tumor, woundsite or disease site. The tumor, wound or disease site may be localizedor systemic in a patient. Targeted delivery may be achieved byrecognition, binding or affinity of a particular receptor or othermolecule that is associated with the tumor, wound or disease by thenanoparticle. The nanoparticles may self-assemble around a therapeutic.The therapeutic may be hydrophobic or hydrophilic. For example, drug andtherapeutic delivery vehicles comprising polypeptides described hereinthat respond to a pH stimulus or temperature change at themicroenvironment of the biological site, may be triggered to release achemotherapeutic cargo in the biological site. The ability to fine tunethe pH responsiveness of drug and therapeutic delivery vehicles may beachieved by selecting a polypeptide having amino acid residues in themotifs which have appropriate pKa values, such that they change theirionization state at relevant pH values.

Polypeptides described herein may be bioactive, or may lose bioactivityor become bioactive after delivery to a biological site. Bioactiverefers to the ability to have biological activity at a biological site,and may include the ability to induce biological effects, therapeuticactivity, or a combination thereof.

Fusion proteins may be formed which include an environmentallyresponsive polypeptide operably connected to a polypeptide of interest.Fusion proteins may be generated by generating a polynucleotide whichincludes a polynucleotide encoding an environmentally responsivepolypeptide operably connected to a polynucleotide encoding apolypeptide of interest and expressing a polypeptide from thepolynucleotides. The expressed polypeptide contains the environmentallyresponsive polypeptide connected or fused to the polypeptide ofinterest. Optionally a linker sequence may be included between thepolynucleotides and fused polypeptides. The linker sequence may be atleast 1, least 2, least 3, least 4, least 5, least 6, least 7, least 8,least 9, least 10, least 15 amino acids and less than 200, 150, 100, 75,50, 40, 30 or 20 amino acids. Expression may be carried out, for examplein a bacterial, yeast or mammalian cell, with the appropriate promotersequence. Fusion proteins may also be generated by chemical synthesis,or by chemically attaching naturally or chemically synthesized peptidesand polypeptides together.

The environmentally sensitive polypeptide may also be chemicallyconjugated to molecules such as therapeutics, carbohydrates, syntheticpolymers, polynucleotides and oligonucleotides, including DNA, RNA, aswell chemically synthesized small molecules.

The present disclosure is not limited in its application to the detailsof construction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The inventiondescribed in the present disclosure is capable of other embodiments andof being practiced or of being carried out in various ways. Also, thephraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting.

The use of “including,” “comprising,” “having,” “containing,”“involving,” and variations thereof herein, is meant to encompass theitems listed thereafter and equivalents thereof as well as additionalitems. For the purposes of promoting an understanding of the principlesof the present disclosure, reference will now be made to preferredembodiments and specific language will be used to describe the same.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. at least one) of the grammatical object of the article.By way of example, “an element” means at least one element and caninclude more than one element.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, andin some embodiments ±0.1% from the specified amount, as such variationsare appropriate to perform the disclosed methods.

Examples Materials and Methods

Gene synthesis of EIPs: EIPs can be synthesized by standard molecularbiology techniques suitable for the synthesis of genes encodingrepetitive protein-polymers, some of which have been previouslydescribed (see, e.g., McDaniel, J. R. et al. (2010) Biomacromolecules10.1021/bm901387t; Meyer, D. E. et al. (2002) Biomacromolecules3:357-367). Many of the EIP genes herein reported were synthesized by anovel method recently developed by the inventors, which are describedelsewhere herein. Regardless of the gene synthesis method used for theirconstruction, the DNA sequence of all of the genes encoding EIPprotein-polymers characterized herein were verified by direct DNAsequencing (Eton Bioscience Inc., NC, USA). Before expression, anN-terminal leader sequence encoding for Met-Ser-Lys-Gly-Pro (SEQ ID NO:62) and a C-terminal His-tag tail encoding for His-His-His-His-His-His-Y(SEQ ID NO: 63) were incorporated into the genes, unless indicated. Theproperties of the compositions subject to the present disclosure areindependent of the aforementioned leader and trailer sequences; however,the incorporation of a His-tag sequence typically results in asignificant increase in the transition temperature of polypeptides,particularly those of low molecular weight (e.g., <20 KDa).

Expression and Characterization of EIPs: Before large-scale expression,starter cultures (3-5 mL) of TB media supplemented with 100 μg/mLampicillin were inoculated with transformed cells from DMSO stocksstored at −80° C., and incubated overnight at 37° C. while shaking at250 rpm. The starter cultures were then centrifuged at 3000 g for 2 min,and resuspended in 1 mL of fresh TB medium. Expression cultures (4 Lflasks containing 1 L of TB media with 100 μg/mL ampicillin) wereinoculated with the resuspended starter culture and incubated at 37° C.with shaking at 200 rpm. After 6-7 h of growth, expression was inducedby the addition of IPTG to a final concentration of 1 mM. The cells wereharvested 24 h after inoculation, and purified by inverse transitioncycling (ITC) as previously described, or, in a limited number of cases,by His-tag purification following the instructions of the manufacturer(Pierce, USA) (see, e.g., Christensen, T. et al. (2009) Protein Science18:1377-1387). To characterize the inverse transition temperature ofEIPs, the optical density of EIP solutions (25-50 μM), prepared in PBSor PBS supplemented with NaCl and/or 8M Urea as indicated, was monitoredat a wavelength of 350 nm (OD₃₅₀) as a function of temperature, at aheating rate of 1° C. min⁻¹, on a Cary 300 UV-visible spectrophotometerequipped with a multicell thermoelectric temperature controller (VarianInstruments, Walnut Creek, Calif.). The derivative of the opticaldensity with respect to temperature was numerically calculated, and theTt was defined as the temperature at the maximum of the turbiditygradient.

Bioinformatics Studies: The amino acid sequence of different elastomericand non-elastomeric proteins was retrieved as FASTA (.txt) filed fromthe National Center for Biotechnology Information protein database.In-house custom methods were implemented in MATLAB software (MathWorks,Natick, Mass.) to map the location of elastin-like polypeptide motifs(e.g., VPGXG (SEQ ID NO: 3), IPGXG (SEQ ID NO: 5)) among the sequence ofthese proteins, the occurrence of generalized P(X_(n))G motifs (where nindicated the number of X residues separating a given Proline andGlycine residue, and varies as 1≦n≦5), the hydrophobicity profiles ofamino acids associated with residues participating or surrounding thesemotifs, the distance between them, and the precise location of Glycineresidues in relation to Glycine residues participating in P(X_(n))Gmotifs. Two different hydrophobicity scales were considered in theseanalyses, the scale proposed by Kyte and Doolittle and Urry et al. (see,e.g., Kyte, J. et al. (1982) J. Mol. Biol. 157:105-132; Urry D. W.(1992) supra).

TABLE 1 Sequence information of the different polypeptides used in thebioinformatics studies. Accession Protein Species Number (GI) ElastinHomo sapiens 182021 Elastin Bos taurus 28461173 Elastin Mus musculus31542606 Elastin Rattus norvegicus 55715827 Elastin Macaca mulatta13182892 Elastin b Danio rerio (Zebrafish) 114326248 Elastin a Daniorerio (Zebrafish) 121583675 Alpha-1 Collagen Type I Homo sapiens 553615Collagen Type III alpha 1 Homo sapiens 4502951 Collagen Type II alpha 1Homo sapiens 111118974 Collagen Type X alpha 1 Homo sapiens 120659966Collagen Type VIII alpha 2 Homo sapiens 32964830 Fibrillin 1 Homosapiens 46559358 Dragline silk fibroin Nephila clavipes 159714 (Spidroin2) flagelliform silk protein Nephila clavipes 2833649 Fibulin 5precursor Homo sapiens 19743803 Fibrillin Homo sapiens 1335064 ResilinIsoform B Drosophila melanogaster 45552671 Resilin Isoform A Drosophilamelanogaster 7302880 High molecular weight Elymus alashanicus 84181091gluten subunit Titin Homo sapiens 1212992 Fibroin-3 (ADF-3) AraneusDiadematus 1263287 Protein PRQFV-amide Aplysia californica 74842069FMRFamide-related Lymnaea stagnalis 1169643 neuropeptides Transcriptionelongation Schizosaccharomyces pombe 74581925 factor spt5

Gene synthesis: The phase transition biopolymers described in thepresent invention were synthesized by standard molecular biologytechniques suitable for the synthesis and expression of genes encodingrepetitive protein-polymers. The genes encoding for the biopolymersherein reported were synthesized by three different methods, describedbelow.

1. All genes, unless indicated, were synthesized using OERCA. Briefly,single stranded DNA sequences were designed encoding for 1 to 5 copiesof the amino acid motif of interest—depending on the motif length—,which were then circularized using a ligase. The circular DNA wasamplified and extended using primers specific for the 5′ and 3′ ends ofthe linear DNA using a polymerase with strand displacement activity in aPCR-type reaction that resulted in gene polymerization by two means:first, by way of rolling circle amplification, and second, by overlapelongation of the extended genes that had rolled from the circle. Thisresulted in a library containing oligomers with various numbers ofrepeats of the starting monomer unit (that already included 1-5 copiesof the motif of interest), which were blunt ligated into a modifiedpET25 vector and transformed into BL21 cells. Clones having genes ofvarious sizes and that were inserted in the correct orientation werescreened, by way of colony PCR and direct DNA sequencing (EtonBioscience Inc., NC, USA). DMSO stocks of all clones that harbored onegene that encoded for any number of repeats of the motif of interestwere prepared.

2. Genes encoding for biopolymers with repeat units longer than 50 aminoacids and with randomized composition were purchased from Mr. Gene andcloned into a modified pET24 vector for expression in E. coli. Weprepared DMSO stocks of the clones that harbored the gene of interest.

3. Genes encoding for biopolymers with the following motifs weresynthesized by Pre-RDL as recently described by the inventors [McDanielet al. 2010]: GRGDSPYQ (SEQ ID NO: 48), GRGNSPYG (SEQ ID NO: 49), LGAPVG(SEQ ID NO: 25), AVPGVGAVPGVGAVPGVGAVPGVGAVPGVGCVPGVG (SEQ ID NO: 64),and VPAGVGVPAGVGVPAGVGVPAGVGVPAGVGVPCGVG (SEQ ID NO: 65). Briefly, wedesigned oligonucleotides for the sense and antisense strands of genesencoding for 1-5 copies of the motifs of interest, which annealedleaving a 3′ GG overhang on the sense strand and a 3′ CC overhang on theantisense stand to allow for concatemerization. The concatemers wereligated into a modified pET24 vector and transformed into E. coli, andthe resulting colonies were screened by colony PCR and direct DNAsequencing (Eton Bioscience Inc., NC, USA). We prepared DMSO stocks ofall clones that harbored one gene that encoded for any number of repeatsof the motif of interest.

Before expression, an N-terminal leader sequence encoding forMet-Ser-Lys-Gly-Pro (SEQ ID NO: 62) and a C-terminal His-tag tailencoding for His-His-His-His-His-His-Y (SEQ ID NO: 63) were incorporatedinto the genes, except for the genes synthesized by (2) and (3), whichlacked the His-tag sequence. The properties of the compositions subjectof the present invention are independent of the aforementioned leaderand trailer sequences; however, the incorporation of a His-tag sequencetypically results in a significant increase in the transitiontemperature of polypeptides, particularly those of low molecular weight(e.g., <20 KDa).

Expression and characterization of EIPs. Before large-scale expression,starter cultures (3-5 mL) of TB media supplemented with 100 μg/mLampicillin were inoculated with transformed cells from DMSO stocksstored at −80° C., and incubated overnight at 37° C. while shaking at250 rpm. The starter cultures were then centrifuged at 3000 g for 2 min,and resuspended in 1 mL of fresh TB medium. Expression cultures (4 Lflasks containing 1 L of TB media with 100 μg/mL ampicillin) wereinoculated with the resuspended starter culture and incubated at 37° C.with shaking at 200 rpm. After 6-7 h of growth, expression was inducedby the addition of IPTG to a final concentration of 1 mM. The cells wereharvested 24 h after inoculation, and purified by inverse transitioncycling (ITC) as previously described (Christensen et al. 2009). Tocharacterize the inverse transition temperature of phase transitionbiopolymers, the optical density of biopolymer solutions (25-50 μM),prepared in PBS or PBS supplemented with NaCl and/or 1-8M Urea asindicated, was monitored at a wavelength of 350 nm (OD350) as a functionof temperature, at a heating rate of 1° C. min-1, on a Cary 300UV-visible spectrophotometer equipped with a multicell thermoelectrictemperature controller (Varian Instruments, Walnut Creek, Calif.). Thederivative of the optical density with respect to temperature wasnumerically calculated, and the Tt was defined as the temperature at themaximum of the turbidity gradient.

Bioinformatics studies. The amino acid sequence of different Pro- andGly-rich proteins was retrieved as FASTA (.txt) files from the NationalCenter for Biotechnology Information protein database. In-house custommethods were implemented in MATLAB software (MathWorks, Natick, Mass.)to map the location of elastin-like polypeptide motifs (e.g., VPGXG (SEQID NO: 3), IPGXG (SEQ ID NO: 5)) among the sequence of these proteins,the occurrence of generalized P(X_(n))G motifs (where n indicates thenumber of X resides separating a given Proline and Glycine reside, andvaries as 1≦n≦5), the hydrophobicity profiles of amino acids associatedwith residues participating or surrounding these motifs, the distancebetween them, and the precise location of Glycine residues in relationto Glycine residues participating in P(X_(n))G motifs. Two differenthydrophobicity scales were considered in these analyses, the scaleproposed by Kyte and Doolittle (1982) and the scale proposed by Urry etal. 1992.

Results

Simple bioinformatics methods were developed and implemented tovisualize and quantify minima functional motifs in elastomeric proteins.FIG. 1 shows a digital map of the distribution and conservation of thecanonical ELP motif VPGXG (SEQ ID NO: 3) along the sequence of elastinfrom various species. Relatively poor sequence conservation and lowsequence coverage were observed. A minima functional P(X_(n))G motif wasmapped, which encompasses multiple potential arrangement of Proline andGlycine residues, among elastin sequences. The results showed asurprisingly high degree of sequence conservation and sequence coverage,mostly corresponding to PG units (i.e., n=0) (FIG. 1C). A similar mapwas drawn from other elastomeric and non-elastomeric proteins whereProline and Glycine residues occur with high frequency. The occurrenceof P(X_(n))G motifs other than the one observed in elastin sequences,particularly those where n=1 and n=4, were observed (see FIG. 2).Moreover, the distribution of these residues was random, as contrastingmaps were observed for P(X_(n))G and G(X_(n))P motifs (see FIG. 2).These maps also uncovered a potential role for Gly residues closelypositioned surrounding the P(X_(n))G motif, as observed when comparingthe elastomeric flagelliform silk (FIG. 2A), where Gly usually occursone residue before Proline, with elastin, gluten or resilin (FIG. 2B-D),where Gly occurs two or more positions before Proline. There was alsoevidence of a high abundance of a PX₄G motif in resilin, but no evidencefor PX₂G and PX₃G motifs was observed (see FIG. 3). Only silk proteinshad a large percentage of GPX_(n) motifs.

Elastomeric proteins display different properties and several mechanismsmay be responsible for such differences; one such mechanism may relateto protein hydrophobicity. Elastomeric proteins, particularly elastin,may include an association of hydrophobic and hydrophilic(cross-linking) domains. A more detailed profiling of the hydrophobicityof key residues participating in the identified PX_(n)G motifs wasconducted. The hydrophobicity of these residues among elastin proteins,showed a distribution of hydropathy indices, which remained largelyhydrophobic for bovine and Homo sapiens, but extended into morehydrophilic values for elastin proteins from Zebrafish and Mus musculus(see FIG. 4). Different evolutionary constraints experienced by thesespecies may have resulted in the selection of amino acids coveringvarious regions of a space of sequences all ascribing to a generalPX_(n)G motif. Evolutionary bias toward the localization of Gly residuestwo positions before the Pro (Pm2; Proline minus 2 residues) and one(Gp1; Gly plus 2 residues) or two (Gp2; Gly plus 2 residues) positionsafter the Gly in the PX_(n)G motif was observed, along with a very lowfrequency of Gly residues occurring one position before the Pro (Pm1;Proline minus 1 residue). The abundant PX₁G motif in collagen (FIG. 3B),however, occurs almost entirely with Gly at Pm1, with no other positionsurrounding the motif being biased toward any particular residue orhydropathy (see FIG. 5). Similar analyses were performed on a largerpool of elastomeric proteins and amino acids covering a broader range ofhydropathy indices were explored; a similar bias in the distribution ofGly residues was found. For instance, resilin and gluten PX₀G motifs aresurrounded primarily by hydrophilic residues, such that the selection ofhydrophobic amino acids in these positions may not be a prerequisite forthe elastic behavior of these proteins. Similarly, the overallhydropathy of Dragline silk and Flagelliform silk are almost identical,being −0.40 and 0.37 (Kyte-Doolittle scale), respectively. Thehydrophobicity of the residues surrounding the abundant PX₀G motif inDragline silk and Flagelliform silk (i.e., Pm2, Pm1, Gp1 and Gp2 in FIG.5) show an average hydropathy for the neighboring residues of −0.16 and−1.2, respectively. Resilin shows an abundant (FIG. 3B) highly conservedPX₄G motif populated by hydrophilic residues (FIG. 7); the conservedPX₄G motif is a continuous motif not abundant in related elastomericproteins (FIG. 3 A-B; FIG. 7).

The results revealed that distribution of Pro and Gly residues may beresponsible for the elasticity and/or environmental responsivenessdisplayed by these proteins. Collagen, although not elastomeric, isthermoresponsive and presents a large number of both PX₀G and PX₁Gmotifs, and has a much larger Pro content than the proteins analyzedherein, particularly Pro residues at Pm1 and Pm2 at PX₀G and PX₁Gmotifs, respectively. The PX₁G motifs have relatively low abundance inmost elastomeric proteins described.

The canonical elastin-like motif includes PX₀G, where only the X residuein the pentapeptide motif VPGXG (SEQ ID NO: 3) was previously believedto be able to accept amino acids of any hydrophobicity. However,bioinformatic studies showed the occurrence of PX₀G motifs with at least4 positions surrounding the PG dipeptide covering a broad spectrum ofhydropathy indices; indeed, there was no evidence for larger sequencediversity at Gp1 (equivalent to X in VPGXG; SEQ ID NO: 3) among elastinsequences and other elastomeric proteins (FIGS. 4 and 5). Therefore, tofurther test the hypothesis, a large data set of polypeptides werecreated incorporating PG, PX₁G, PX₁X₂G (SEQ ID NO: 16) and PX₁X₂X₃X₄G(SEQ ID NO: 66) motifs while varying the residues that surround orconstitute the P(X_(n))G motifs, and engineering Gly residues atdifferent positions around the P(X_(n))G motif to study the role of Glyat these positions, as suggested by the bioinformatics study below inTable 2.

TABLE 2Minima functional environmentally sensitive motifs that were reverseengineered from the sequence of a variety of elastomeric and non-elastomeric proteins(see Table 2), and the experimental evidence from corresponding EIP motifs demonstrated todisplay elasticity and/or environmental sensitivity. EIPs of various lengths for a singlerepeat unit were generated to study the role of molecular weight on their behavior.Equivalent EIP constructs Minimum functional motif Repeat unit*Z and X values Gp2, Pm2, Pm1 P(X₀)G AVPGVG Z₁ = A, Z₂ = V, Z₃ = V, Z₄ =G Gp2 = G Z₁Z₂ PGZ₃Z₄ (SEQ ID NO: 8) VAPGVG Z₁ = V, Z₂ = A, Z₃ = V, Z₄ =G Gp2 = G (SEQ ID NO: 67) GVPGAV Z₁ = G, Z₂ = V, Z₃ = A, Z₄ = V Pm2 = G(SEQ ID NO: 68) GVPGVA Z₁ = G, Z₂ = V, Z₃ = V, Z₄ = A Pm2 = G(SEQ ID NO: 69) TVPGVG Z₁ = T, Z₂ = V, Z₃ = V, Z₄ = G Gp2 = G(SEQ ID NO: 70) TVPGAG Z₁ = T, Z₂ = V, Z₃ = A, Z₄ = G Gp2 = G(SEQ ID NO: 71) GAPGVG Z₁ = G, Z₂ = A, Z₃ = V, Z₄ = G Gp2 = G(SEQ ID NO: 72) & Pm2 = G AVPGVA Z₁ = A, Z₂ = V, Z₃ = V, Z₄ = A Gp2< >G(SEQ ID NO: 73) & Pm2< >G GAPGGG Z₁ = G, Z₂ = A, Z₃ = G, Z₄ = G Gp2 = G(SEQ ID NO: 74) & Pm2 = G GAPGAG Z₁ = G, Z₂ = A, Z₃ = A, Z₄ = G Gp2 = G(SEQ ID NO: 75) & Pm2 = G G G PGAG Z₁ = G, Z₂ = G, Z₃ = A, Z₄ = G Gp2 =G (SEQ ID NO: 76) & Pm2 = G & Pm1 = G P(X₀)G GVPAGVG Z₁ = G, Z₂ = V, X =A, Z₃ = V, Z₄ = G Gp2 = G Z₁Z₂ PX 1 GZ₃Z₄ (SEQ ID NO: 77) & Pm2 = G VGPVGVG Z₁ = V, Z₂ = G, X = V, Z₃ = V, Z₄ = G Gp2 = G (SEQ ID NO: 78) &Pm1 = G GVPTGVG Z₁ = G, Z₂ = V, X = T, Z₃ = V, Z₄ = G Gp2 = G(SEQ ID NO: 79) & Pm2 = G GAPVGVG Z₁ = G, Z₂ = A, X = V, Z₃ = V, Z₄ = GGp2 = G (SEQ ID NO: 80) & Pm2 = G VAPVG VA Z₁ = V, Z₂ = A, X = V, Z₃ =V, Z₄ = A Gp2< >G (SEQ ID NO: 81) & Pm2< >G GAPFGFA Z₁ = G,Z₂ = A, X =F, Z₃ = F, Z₄ = A Pm2 = G^(Ψ) (SEQ ID NO: 82) AIPMGAG Z₁ = A, Z₂ =I, X = M, Z₃ = A, Z₄ = G Gp2 = G^(Ψ) (SEQ ID NO: 83) GFPTGGL Z₁ =G, Z₂ = F, X = T, Z₃ = G, Z₄ = L Pm2 = G^(Ψ) (SEQ ID NO: 84) LAPFGMGZ₁ = L, Z₂ = A, X = F, Z₃ = M, Z₄ = G Gp2 = G^(Ψ) (SEQ ID NO: 85)GLPAGMG Z₁ = G, Z₂ = L, X = A, Z₃ = M, Z₄ = G Gp2 = G (SEQ ID NO: 86) &Pm2 = G^(Ψ) P(X2)G GVPAVG V Z₁ = G, Z₂ = V, X₁ = A, X₂ = V, Z₃ = V Pm2 =G Z₁Z₂ PX 1 X 2 GZ₃ (SEQ ID NO: 87) GVPHVG V Z₁ = G, Z₂ = V, X₁ =H, X₂ = V, Z₃ = V Pm2 = G (SEQ ID NO: 88) VGPAVG V Z₁ = V, Z₂ = G, X₁ =A, X₂ = V, Z₃ = V  Gp2 = G (SEQ ID NO: 89) & Pm1 = G VTPAVG V Z₁ =V, Z₂ = T, X₁ = A, X₂ = V, Z₃ = V Gp2< >G (SEQ ID NO: 90) & Pm2< >GP(X4)G GVPSALYGVG Z₁ = G, Z₂ = V, X₁ = S, X₂ = A, Gp2 = G Z₁Z₂ PX 1 X 2X 3 X 4 GZ₃Z₄ (SEQ ID NO: 91) X₃ = L, X₄ = Y, Z₃ = V, Z₄ = G & Pm2 = GGVPSDDYGQG Z₁ = G, Z₂ = V, X₁ = S, X₂ = D, Gp2 = G (SEQ ID NO: 92) X₃ =D, X₄ = Y, Z₃ = Q, Z₄ = G & Pm2 = G ** GVPSDDYGVG Z₁ = G, Z₂ = V, X₁ =S, X₂ = D, Gp2 = G (SEQ ID NO: 93) X₃ = D, X₄ = Y, Z₃ = V, Z₄ = G &Pm2 = G *Letters underlined are not part of the EIP repeat unit in theconstructed polypeptides, but correspond to a residue in the n?‘repeatunit as it is presented in tandem. ^(Ψ)The functionality of these motifswas assessed as part of a single polypeptide generated by using a methodthat randomized the selection of amino acids for 5 hexapeptidesfollowing a Z₁Z₂PX₁GZ₃ (SEQ ID NO: 22) motif, which were then repeatedin tandem. The design principle entailed having a G residue at eitherPm2 or Gp2. A normal distribution was used for selection of residueswith a target Hi of 2 for both X and Z residues and a standard deviationof 1.5. The sequence of such randomized polypeptide is:(GAPFGFAIPMGAGFPTGGLAPFGMGLPAGM)_(n) ((SEQ ID NO: 12)_(n)). **Theseelastic sequences display thermal stability and solubility due to thelarge contribution of the hydrophilic aspartate residues confined withinthe PXXXXG (SEQ ID NO: 94) motif to the inverse transition temperatureof these sequences.

The environmental responsiveness of the motifs in Table 2 (see FIGS.8-16) was characterized and demonstrated the robustness of the P(X_(n))Gmotifs to confer environmental sensitivity and elasticity topolypeptides, and the possibility to engineer flexible functionalZ_(m)P(X_(n))GZ_(k) (SEQ ID NO: 95) motifs, wherein Z_(m) and Z_(k) areeach amino acids of any hydrophobicity which surround the P(X_(n))Gfunctional unit and n, m, k≧4. For simplicity and clarity, the data inFIG. 8 through 16 is presented by grouping all the polypeptidesdescribed by a common Z_(m)P(X_(n))GZ_(k) (SEQ ID NO: 95) motif.

The characterization of retro-EIPs, in which the sequence of the motifwas backbone reversed (see Table 3 below) while regenerating a PX_(n)Gmotif typically with different n value, further demonstrated therobustness of these motifs since environmentally sensitivity wasmaintained in all cases (see FIG. 17). Backbone reversal of the motifAPGVG (Table 2) would result in an EIP with a PX₁X₂X₃G (SEQ ID NO: 96)motif, which constitutes an additional minima functional motifZ_(m)P(X_(n))GZ_(k) where n=3.

TABLE 3Backbone-reversed -retro- EIPs. The sequence of an EIP motif as read fromthe N- to C-terminus was reversed so that the sequence was identical when read fromthe C- to N- terminus. In addition, whenever a Gly residue occurred at Pml as a resultof backbone reversal, a modified retro-motif was synthesized in which an Ala orThr residue was substituted for Gly, in order to study the effect of Gly at Pm1.Modified Motif Retro-motif retro-motif* Minimum Minimum Minimum unit EIPunit EIP unit EIP PX₀G VPGVG PX₁G VGPVG PX₁G V A PVG (SEQ ID NO: 97)(SEQ ID NO: 98) PX₀G VAPGVG PX₂G VGPAVG PX₂G V T PAVG (SEQ ID NO: 67)(SEQ ID NO: 17) PX₁G VPAGVG PX₁G VGAPVG — (SEQ ID NO: 33)(SEQ ID NO: 24) PX2G VPAVG PX1G VAPVG — (SEQ ID NO: 6) (SEQ ID NO: 27)*The modified (substituted or inserted) residue from the originalretro-motif is shown underlined.

The data showed that biased selection of amino acids in key motifsobserved in repetitive proteins is representative of the evolutionaryconstraints experienced by different species. A surprising bias in thelocalization of Gly residues at Pm2 and Gp2 (at either position orsimultaneously occurring for a given PX_(n)G repeat unit) surroundingPX_(n)G motifs in elastomeric proteins (see FIGS. 4-6) is not aprerequisite for the reversible phase transition behavior displayed byelastomeric-inspired polypeptides, as demonstrated by EIPs with motifsVAPVG (SEQ ID NO: 27) (FIG. 13) and APGVG (SEQ ID NO: 99) (FIG. 11).This observation reinforces the possibility of constructing trulygeneral motifs as described in Table 2, where Z residues do not have tobe restricted to Gly at either Pm1 or GP2, while still preserving theenvironmental sensitivity and elasticity observed in otherZ_(m)P(X_(l))GZ_(k) (SEQ ID NO: 95) and Z_(m)PGZ_(k) (SEQ ID NO: 100)motifs. In addition, the demonstration of the environmental sensitivityof a randomized EIP with repeat unit

(SEQ ID NO: 101)Z₁Z₂PX₁GZ₂Z₃Z₄PX₂GZ₆Z₇Z₈PX₃GZ₉Z₁₀Z₁₁PX₄GZ₁₂Z₁₃Z₁₄PX₅GZ₁₅displaying a minima functional PX₁G motif constitutes a step further inthe design of “smart” protein-polymers (see FIG. 12A).

Regarding the role of neighboring Gly residues in the functionality ofthe P(X_(n))G motif, FIG. 18 demonstrates that Gly at Pm1 of a P(X_(n))Gfacilitates self-assembly, presumably by making the packing of thepolypeptide chains more efficient due to reduced steric hinderanceprovided by the side-chain motifs having a more hydrophobic residue atPm1, and by promoting the formation of stable non-reversible fibrillarstructures rather than driving the self-assembly of fractal structures.Sheparavych et al. have shown that for other self-assembling peptides,the disruption of the peptide secondary structure, particularly theirhelical conformation, is key to the self-assembly process in thisfractal manner (Sheparavych, R. et al. (2009) Biomacromolecules10:1955-1961). Interestingly, the bioinformatics analyses only pointedto the occurrence of Gly at Pm1 for collagen and silks, and in bothcases, abundant secondary and tertiary structures are present, which isin agreement with the finding that Gly at Pm1 increases the propensityof these motifs to drive irreversible phase separation and disruption ofelasticity above a critical temperature. In addition, it has also beenshown that other interesting self-assembly properties for PX₁X₂G motifs,as shown in FIG. 19, and as reported in the literature for the ELP withsequence VPAVG (SEQ ID NO: 6), which also carries a PX₁X₂G (SEQ ID NO:102) motif (see, e.g, Bessa, P. C. et al. (2009) supra).

An additional feature of the EIP motifs disclosed in the presentdisclosure is the possibility to exploit their environmental sensitivityfor their efficient purification, in an analogous manner to thepurification schemes in use for the preparation of ELPs. It has beenobserved by the inventors that even those EIPs that displayheat-irreversible phase separation, typically display reversible phaseseparation in response to changes in buffer ionic strength, and thisproperty has been exploited for their high yield purification. However,the expression (in E. coli) and purification of EIPs with Gly at Pm1 issomewhat more difficult compared with the ease of purification of otherEIPS, primarily due to their tendency to form insoluble aggregates andform inclusion bodies during protein expression. Interestingly, althoughGly at Pm1 may drive the heat-irreversible phase separation, it does notnecessarily disrupt the reversible phase separation when using anorthoganol stimulus, such as buffer ionic strength.

It was reasoned that EIPs displaying heat-irreversible inverse phasetransition can be engineered as elastomeric or non-elastomeric materialsif cross-linked below or above the threshold temperature for theheat-irreversible phase separation of a given EIP. This is in accordancewith Urry's observation that potential elastomers display reversibleaggregation, whereas non-elastomeric polypeptides display irreversibleaggregations (U.S. Pat. No. 5,250,516). Noteworthy, the cross-linkingconditions could be readily adjusted to tune the transition temperature(Tt) of the polypeptide to a temperature range below the thresholdtemperature, by exploiting the sensitivity of EIPs to changes in bufferionic strength. In addition, it has also been observed by the inventorsthat such threshold temperature is typically above body temperature, sothat devices composed of EIPs displaying heat-irreversible phaseseparation should not be compromised upon implantation, especially ifcross-linked. In addition, heat-irreversible phase separation ornon-elastomeric behavior may be favored by engineering Gly residues atPm1 or PX_(n)G minima functional motifs that would promote the formationof stable perhaps crystallizable structures above a thresholdtemperature above the Tt. A recent study by Chen and Guan supports theidea that the localization of Pro and Gly residues in the canonical ELPmotif VPGXG (SEQ ID NO: 3) serves a role in disrupting secondarystructures that would otherwise prevent the highly elastic behavior anddynamic nature of these polypeptides, and rather promote the formationof random coils or dynamic low stability conformations as those based onβ-turns and Polyproline-II conformations (Chen, Y. et al. (2009) J. Am.Chem. Soc. DOI:1021/ja9104446). Therefore, the ability to engineer thedevelopment of such ordered structures in unordered EIPs may provideadditional means to control the elasticity of a protein-polymer in astimuli-controlled manner.

In addition, the present disclosure describes the thermally responsivebehavior of resilin-inspired polypeptides. Resilin-like polypeptideswith the 11 residue repeat AQTPSSQYGAP (SEQ ID NO: 103) have beenregarded in the literature as unordered polypeptides and were notreported to show thermally responsive behavior. A highly conserved YGAP(SEQ ID NO: 104) motif was suspected to be required for the properties,namely elasticity and resilience, of this polypeptide (see, e.g., Nairn,K. M. et al. (2008) supra). In contrast, the present disclosureindentifies a functional PX₄G motif in resilin, which can also beidentified in the repeat unit AQTPSSQYGAP (SEQ ID NO: 103), anddemonstrated its environmental sensitivity (FIG. 16) and the possibilityto generalize the sequence.

The present disclosure demonstrates the possibility to synthesizebioactive EIPs for tissue engineering and regenerative medicineapplications by synthesizing EIPs incorporating the bioactive potentproangiogenic GXXPG (SEQ ID NO: 21) motif found in elastin (see, e.g.,Robinet, A. et al. (2005) J. Cell Science 118:343-356), which displayidentical behavior to nonbioactive conventional elastin-likepolypeptides and thus display remarkable environmental sensitivity andelasticity (FIG. 20). This is the first demonstration of an elastic,environmentally sensitive polypeptide carrying a potent chemokine, sincenon-elasticity of the VAPGVG (SEQ ID NO: 67) hexapeptide has frustratedmajor engineering efforts exploiting the functionality of this bioactivemotif.

A large number of genetically encoded protein-based polymers weresynthesized that span the entire range of Pro-X_(n)-Gly arrangementsobserved in the bioinformatics studies (that is, n=0-4), to assesswhether they retained the phase behavior characteristic of the canonicalVal-Pro-Gly-X-Gly (SEQ ID NO: 3) motif found in tropoelastin. FIG. 21confirms that all these new arrangements of Pro and Gly residues areconducive to “smart” biopolymers with stimuli-responsive, phasetransition behavior analogous to that of tropoelastin. Theseprotein-polymers appear to have similar secondary structure propensitiesas elastin-like polypeptides and display an ensemble of highly dynamicconformers characteristic of other IDPs (FIG. 21C). This excitingfinding significantly relaxes the sequence constraints on thesepolymers, as it allows for the incorporation of a number of short motifswithin the PX_(n)G unit or in the surrounding residues—10 residuesbetween PX_(n)G units are permissible. For instance, a number ofneuroactive proteins present their bioactive sequences as tandem repeatsembedded within PX₄G units. Pro-rich proteins use tandem repeats of PX₄Gmotifs for the presentation of bioactive peptides. The two neuroactiveproteins FARXamide-related neuropeptides and PRQFVamide display tandemrepeats of the bioactive peptides PFLRF (SEQ ID NO: 108) and PRQFV (SEQID NO: 109) embedded within PX₄G motifs. A similar localized region ofPX₄G units was observed with highly conserved X₄ residues in atranscription factor from yeast (SPT5).

It is also possible to identify “smart” biopolymers with three types ofphase behavior marked by three degrees of hysteresis in thereversibility of their thermally-triggered phase transition: i) zero,ii) finite, and iii) heat-sensitive infinite hysteresis (FIG. 21D). The“heat-sensitivity” of the latter category refers to the finding thatthese protein-polymers display zero hysteresis below a criticalthreshold temperature (typically around 40° C. for the polymers hereinsynthesized). Six different environmentally responsive polypeptides(VGAPVG)₃₅, (VGPVG)₃₀, (VGPAVG)₂₀, (VPGAVG)₃₀, (VTPAVG)₂₅, and(TPVAVG)₃₀, (repeats of SEQ ID NOs: 24, 98, 17, 38, 18 and 31respectively) were found to show irreversible phase separation whenheated to 75° C., whereas they displayed reversible phase transitionbehavior if heated below a given threshold temperature. The (TPVAVG)₃₀(SEQ ID NO: 31) polypeptide was purified by exploiting the reversibilityof its phase behavior in response to changes in ionic strength.Environmentally responsive polypeptides with the repeat unit (VTPAVG)(SEQ ID NO: 18) exhibited a very complex phase transition behavior asthey displayed both heat-sensitive infinite hysteresis and finitehysteresis below the threshold temperature. The phase behavior wascharacterized in PBS at a polypeptide concentration of 50 μM.

The heat-sensitive, infinite hysteresis of the environmentallyresponsive polypeptides was found to arise from the emergence of orderedsecondary structures that stabilized the insoluble phase. Whereas anenvironmentally responsive polypeptide that displayed finite hysteresisrapidly recovered its conformational disorder on lowering thetemperature below the phase transition temperature adjusted by itsdegree of hysteresis, environmentally responsive polypeptides withheat-sensitive infinite hysteresis underwent conformational changes thatpersisted on cooling. An environmentally responsive polypeptide thatdisplayed such a large hysteresis did not exhibit observablereversibility below any given temperature threshold, and displayedordering on coacervation. Turbidity and CD data were acquired in waterat a polypeptide concentration of 5 μM.

The emergence of more ordered secondary structures on phase transitionwas identified as a primary factor responsible of the increasing degreeof hysteresis. Despite the respective degree of hysteresis, whichprovides a tool for the biomedical and biotechnological exploitation ofthese materials, the reversible phase transition behavior of allprotein-polymers herein described enabled their purification byrecursive rounds of phase separation.

Having relaxed the constraints on the distribution of Pro and Glyresidues in these protein-polymers, sequence diversity was maximized.The bioinformatics studies underscored the role of overallhydropathicity over local biases in hydrophobicity. Protein-polymerswere designed with a target, average hydropathy, but incorporating aminoacids with a wide distribution of hydropathies. FIG. 22A shows thegeneration of protein-polymers composed of hexapeptide motifs whereinonly one Pro and one Gly residue are fixed and all residues areotherwise randomized. A target hydropathy of 37° C. was selected togenerate a protein-sized biopolymer (240 resides in length) with awidely diverse amino acid composition spanning the entire range ofhydropathies (FIG. 22B).

The primary structure of the randomized environmentally responsivepolypeptide reported in FIG. 22 was of the form (Z-Z-P—X-G-Z)₄₀ and hadthe following sequence:SKGPGVPAGHRYPIGGGQPHGKGCPDGVFRPVGLGAPYGHGAPNGMHRPLGIGKPRGHMYPKGQGQPMGHLVPDGVGFPRGRKKPVGVGKPIGNGHPIGARTPLGYGMPDGVGMPMGLFLPNGHGAPHGQGYPAGKLIPKGKGHPFGKGRPLGAGRPTGFKMPKGLGKPMGVGQPQGHFVPFGLGQPTGQGAPRGGSQPAGLGHPLGAGAPAGRCHPYGMGVPRGLAMPRGHGQPRGVGYPKGHGWP) (SEQ ID NO: 105). The amino acid sequence included theN terminal peptide SKGP (SEQ ID NO: 106) and the C-terminal tripeptideGWP. The Z and X residues were randomized using a normal distributionwith a mean hydropathy of 37° C. and a standard deviation of 50° C.—inorder to ensure large sequence diversity.

This protein-polymer behaved as an IDP (FIG. 22C) and displayed phasetransition behavior (FIG. 22D). Surprisingly, the target hydropathy wasa good predictor of the temperature at which the biopolymer underwentphase separation (˜40° C.). This protein-polymer lacked any repeatingmotif (unlike tropoelastin or resilin), and Pro-X-Gly units were quiterare in all proteins that we analyzed. A “random” biopolymer wasdesigned the size of a short protein-domain (30 amino acids in length).Polymers of this domain displayed phase transition behavior (FIG. 24).Although Gly enrichment observed in the surroundings of thePro-X_(n)-Gly units for most non-fibrillar Pro and Gly-rich proteins wasincorporated, motifs were identified that demonstrate that such Glyenrichment is not necessary for the design of “smart” biopolymers withfully reversible phase transition behavior (FIG. 25).

Gly has a role in modulating the assembly behavior of IDPs. The phasebehavior of two protein-polymers were studied wherein Gly was placedpreceding a Pro-Gly and a Pro-X-X-Gly unit, and their behavior comparedwith those of identical motifs where this Gly residue was mutated.Positioning of a Gly residue N-terminal to a Pro-X_(n)-Gly motifenhances the propensity for coacervation—by decreasing the transitiontemperature (FIG. 22E-F)—, promotes the formation of irreversibleaggregates (FIG. 22E) and leads to changes in assembly behavior (insetof FIG. 22F). This Gly-induced instability was also evidenced on theinsoluble expression of these protein-polymers (data not shown), despitetheir high solubility in PBS once purified. These results underline therole of Gly as a potent modulator of the assembly of IDPs such that thismodulatory role may be exploited for the synthesis of “smart”biopolymers that reproduce the assembly behavior and/or mechanicalproperties of collagen and silks.

The complex phase behaviors indicate a relationship between the syntaxof the protein-polymer, its secondary structure and its phase behavior.These structure-function relationships are characteristic of foldedproteins. IDPs exhibit highly flexible backbone conformations, but areunlikely to be true random coils. To demonstrate that the polypeptideconformation—as opposed to composition—exerts a potent modulatory rolethat cannot be explained by a simple random coil model of the disorderedstate of these polymers, the effect of backbone reversal on phasebehavior was studied. Backbone reversal produces a biopolymer that hasan identical sequence as the parent biopolymer if read from the C- tothe N-terminus, thus having identical hydrophobicity and identicaldistribution of amino acids (FIG. 22G). However, whereas folded proteinsoften lose their structure and function on backbone reversal, the effectof backbone reversal on the function (that is, phase behavior) of Pro-and Gly-rich polymers that are intrinsically disordered was unexpectedlyfound to lead to changes in function (FIG. 21H-J and FIG. 26A), whichresult from changes in the ensemble of conformations that describe thedynamic backbone of these polymers (FIG. 21K and FIG. 26B-D). Thisfinding suggests that these proteins are unlike synthetic polymers thatexist as random coils, and are protein-polymers that are intrinsicallydisordered. The recurrent link between intrinsic disorder and “smart”behavior suggests that a polypeptide with intrinsic disorder displays“smart” behavior.

Environmentally responsive polypeptides with truly protein-likecomplexity may exert a biological function encoded in their sequence.Their syntax may also be compatible with sequences that have definedsecondary structure propensities as these abound in most proteins.Environmentally responsive polypeptides were made with a repeating unitbased on the matrikine motif Gly-X-X-Pro-Gly (SEQ ID NO: 21) (FIG.4A)—encoding bioactive motifs released on cleavage of variousextracellular matrix proteins—, unlike environmentally responsivepolypeptides with a disrupted motif but identical composition and phasebehavior (FIG. 4B), are capable of preventing tumor growth in a mousemodel (FIG. 4C). An environmentally responsive polypeptide inspired inthe matrikine motif GXXPG (SEQ ID NO: 21), SM1-24 (250 μM in PBS), wasfound to prevent the grafting of 1×10⁶HT1080 tumor cells inoculated intothe back of nude mice (FIG. 27). A control polypeptide, SM2-24 (250 μMin PBS), with a disrupted motif but identical phase transition behavior(FIG. 23) had no effect on tumor growth. Tumor volumes were measured 19days after inoculation.

An environmentally responsive polypeptide was synthesized based on thebioactive site of murine Endostatin, and was found to displays aninverse phase transition temperature reminiscent of otherenvironmentally responsive polypeptides with simpler syntax (FIG. 38).The phase transition of a 5 μM solution of mEndo1-6 in PBS (pH 6.4) (A)was accompanied by a decrease in the disorder of the polypeptideconformation (B), as measured by circular dichroism under identicalconditions as in (A).

The diversity of the ensemble of structures observed in ourenvironmentally responsive polypeptides are compatible with proteindomains that have very defined local secondary structure propensities.Environmentally responsive polypeptides were synthesized that werecomposed of the bioactive domains of endostatin from humans and mice,which are well folded protein domains (25-27 amino acids in length) thatretain the potent anti-angiogenic activity of endostatin as isolatedpeptides (FIG. 23D). These environmentally responsive polypeptides thatbehave as IDPs (FIG. 23E) and display “smart” behavior (FIG. 23F). Thisis surprising given the partially folded nature of these domains in thenative protein (FIG. 23D), the existence of significant regions withhigh propensities to fold into α-helices and β-sheets in the polymerizedpolypeptides (FIG. 23G), and the relatively low Pro and Gly content (10%Pro and 14-17% Gly, compared with 20% Pro and 40% Gly in elastin-likepolypeptides). Peptide hormones, which often have similar, localsecondary structure propensities (FIG. 4G) may be designed and formedfrom environmentally responsive polypeptides described herein.

Environmentally responsive polypeptides were also made that showed UCSTbehaviour (FIG. 28). Such polypeptides were found to display reversibleUCST behavior in PBS which behavior may be tuned by polypeptideconcentration and the number of repeating units. (FIG. 29).

Environmentally responsive polypeptides incorporating the peptide drugGRGDSP were found to be bioactive and their bioactivity could beswitched on or off by their phase transition behavior. (FIG. 30; Leftpanel) Whereas increasing concentrations of (GRGDSPYG)-12 (SEQ ID NO:44)₁₂, which has an UCST below 37° C. and is thus soluble, preventedcell adhesion of PC3-luc-C6 cells after a 3 h treatment, (GRGDSPYG)-20(SEQ ID NO: 44)₂₀, which displayed an UCST above 37° C., had almost noeffect on cell adhesion as its concentration increases. (FIG. 30; Rightpanel) These bioactive environmentally responsive polypeptides were nottoxic to the cells as they remained viable when given sufficient time toadhere—perhaps through mechanisms that are not dependent on theintegrins targeted by GRGDSP. Control cultures that matched theexperimental treatment with the maximum concentration of residual Urea((GRGDSPYG)-12 (SEQ ID NO: 44)₁₂) demonstrated that residual Urea (up to0.1 M for the 40 μM samples) did not affect cell adhesion or viability.

The UCST behavior of environmentally responsive polypeptides containingRGD tripeptides exhibited a complex response to buffer ionic strength,wherein small concentrations of salt decreased the UCST (due to chargescreening) and high concentrations had the opposite effect as theyfavored hydrophobic interactions (FIG. 31).

The UCST behavior of environmentally responsive polypeptides wasmodulated by electrostatic interactions between positively andnegatively charged amino acids within the sequence (FIG. 32). At pH 2.0,aspartic acid was fully protonated, which largely increased thehydrophobicity of (GRGDSPYG)-20 ((SEQ ID NO: 44)₂₀), and yet, instead ofobserving an increase in its UCST (as it would be expected), a drasticreduction in the UCST was observed that demonstrates the role ofelectrostatic interactions—here between Arg and Asp residues—inincreasing the UCST of these biopolymers.

The UCST behavior of environmentally responsive polypeptides did notrequire electrostatic interactions (FIG. 33). The aspartic acid inRGD-containing polypeptides was substituted with asparagine and did noteliminate the UCST behavior displayed by these biopolymers. Thesebiopolymers when tested may also have displayed LCST behavior.

The design of RGD-containing environmentally responsive polypeptidesthat display UCST behavior was found to be compatible with multiplearrangements of Pro and Gly residues. A PG dipeptide, instead of a P-Y-Gtripeptide, did not perturb the UCST behavior (FIG. 34). The UCSTbehavior of the polypeptides was tuned by adjusting the hydrophobicityof the residues comprising the repeating unit. Substituting a Glyresidue by a more hydrophilic residue, glutamine, significantly reducedthe UCST of the biopolymer (FIG. 35).

Environmentally responsive polypeptides that contain the peptide drugPHSRN (SEQ ID NO: 107) were also found to display UCST behavior (FIG.36). These biopolymers exhibited reversible phase behavior (left) andwere intrinsically disordered (right). The UCST behavior of thepolypeptides thus arose from residues capable of intermolecular hydrogenbonding (that is, arginine and serine).

Environmentally responsive polypeptides were designed to display complexphase behaviors wherein the biopolymers display both UCST and LCST, andthe LCST is lower than the UCST. (FIG. 37). Environmentally responsivepolypeptides with composite motifs composed of one UCST motif and oneLCST motif would enable the design of biopolymers that display both UCSTand LCST if the LCST is greater than the UCST. Tuning this band-passbehavior will enable the design of environmentally responsivepolypeptides that display phase separation only in a very narrowtemperature window, which would facilitate the manipulation of complexmixtures of environmentally responsive polypeptides, such as inapplications involving multiplexing.

Any patents or publications mentioned in this specification are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication was specifically and individually indicatedto be incorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

1. An environmentally responsive polypeptide comprising at least tenrepeats of an amino acid sequence Z₁Z₂PX₁X₂X₃X₄GZ₃, wherein P isProline, G is Glycine, X₁, X₂, X₃ and X₄ are an amino acid that is notProline or Glycine and X₁, X₂ and X₃ are optionally absent, Z₁, Z₂ and,Z₃ are each an amino acid, and wherein the polypeptide upon stimulationundergoes a conformational change that is accompanied by aggregation. 2.The polypeptide of claim 1, wherein the at least ten repeats areconsecutive.
 3. The polypeptide of claim 1, wherein the polypeptide is apolymer comprising diblocks or triblocks of the amino acid sequence. 4.A fusion protein comprising the polypeptide of claim
 1. 5. A compositioncomprising the polypeptide of claim 1, conjugated to a molecule.
 6. Thecomposition of claim 5, wherein the molecule is selected from anoligonucleotide, a therapeutic, a carbohydrate, a synthetic polymer, ora combination thereof.
 7. The polypeptide of claim 1, further comprisinga spacer sequence between at least two of the at least ten repeats, thespacer sequence comprising from one to twenty-six amino acids.
 8. Thepolypeptide of claim 7, wherein the spacer sequence comprises at leastone proline.
 9. The polypeptide of claim 1, wherein the amino acidsequence comprises a motif selected from the group consisting of GVPXGV,VGPXVG, GZ₂PVGV, VPXVGZ₃, VZ₂PVGZ₃, GVPXGZ₃, Z₁GPXVG, GZ₂PVGZ₃, whereinX is a single amino acid.
 10. The polypeptide of claim 9, comprising acombination of two or more different motifs.
 11. The polypeptide ofclaim 1, wherein the amino acid sequence comprises a motif conveyingLCST transition behavior, a motif conveying UCST transition behavior, ora motif conveying both LCST and UCST transition behavior.
 12. Thepolypeptide of claim 1, wherein the amino acid sequence comprises afirst motif conveying LCST or UCST transition behavior, and wherein theamino acid sequence further comprises a second motif, wherein the secondmotif conveys UCST transition behavior when the first motif conveys LCSTbehavior, and wherein the second motif conveys LCST behavior when thefirst motif conveys UCST behavior, such that the polypeptide displaysboth UCST and LCST transition behavior.
 13. The polypeptide of claim 1,wherein the polypeptide exhibits heat-irreversible phase separation whenexposed to a threshold temperature that is above a lower criticalsolution temperature of the polypeptide, and exhibits reversible phaseseparation below the threshold temperature.
 14. The polypeptide of claim1, wherein the polypeptide exhibits a reversible phase separation inresponse to a first stimulus.
 15. The polypeptide of claim 14, whereinthe polypeptide exhibits an irreversible phase separation in response toa second different stimulus.
 16. A polypeptide comprising the at leastten repeats of the polypeptide of claim 1 as a reverse sequence whenread from C-terminus to the N-terminus.
 17. An environmentallyresponsive polypeptide comprising at least ten repeats of an amino acidsequence Z₁PXGZ₂Z₃Z₄Z₅, wherein P is proline, G is glycine, X is from 0to 4 amino acids that are not proline or glycine, and Z₁, Z₂, Z₃, Z₄ andZ₅ are each an amino acid and Z₂ is optionally absent, wherein the aminoacid sequence comprises (i) an arginine, (ii) serine or an amino acidselected from aspartate and glutamate, or both serine and aspartate orglutamate, and (iii) at least one hydrophobic residue selected fromvaline, leucine, isoleucine, histidine, tyrosine, tryptophan andalanine, wherein the polypeptide upon stimulation undergoes aconformational change that is accompanied by aggregation.
 18. Thepolypeptide of claim 17, wherein X is HSRN, wherein H is histidine, S isserine, R is arginine and N is asparagine.
 19. The polypeptide of claim17, wherein the amino acid sequence comprises an RGD motif, wherein R isarginine, G is glycine and D is aspartate.
 20. The polypeptide of claim17, wherein the amino acid sequence comprises SPXGGRGD or SPXQGRGD,wherein X is a single amino acid.
 21. A environmentally responsivepolypeptide comprising at least ten sequences selected from VGAPVG,LGAPVG, VPSALYGVG, VGPAVG, VTPAVG, VPSDDYGQG, VPSDDYGVG, TPVAVG,VPSTDYGVG, VPAGVG, VPTGVG, VPAGLG, VPHVG, VHPGVG, VPGAVG, VPGVAG, VRPVG,GRGDSPY, GRGDSPH, GRGDSPV, GRGDSPYG, RPLGYDS, RPAGYDS, RPXGYDS, GRGDSYP,GRGDSPYQ, GRGNSPYG, GRGDAPYQ, VPXSRNGG, VPHSRNGG, VPHSRNGL,VPGHSHRDFQPVLHLVALNSPLSGGMRG, HTHQDFQPVLHLVALNTPLSGGMRGIRPGG,FEWTPGWYQPYG or a combination thereof, wherein X is from zero to fouramino acid residues, and wherein the polypeptide upon stimulationundergoes a conformational change that is accompanied by aggregation.22. The polypeptide of claim 21, wherein the at least ten sequences areconsecutive.
 23. The polypeptide of claim 21, further comprising aspacer sequence between at least two of the at least ten sequences. 24.The polypeptide of claim 21, wherein the polypeptide exhibits phaseseparation when exposed to a threshold temperature that is (i) above alower critical solution temperature of the polypeptide, or (ii) below anupper critical solution temperature of the polypeptide, or exhibitsphase separation when exposed to a threshold temperature that is abovethe lower critical solution temperature, and when exposed to a thresholdtemperature that is below the upper critical solution temperature. 25.The polypeptide of claim 24, wherein the phase separation is reversible.26. The polypeptide of claim 24, wherein the phase separation isirreversible.
 27. The polypeptide of claim 21, wherein the at least 10sequences convey LCST or UCST transition behavior, and wherein thepolypeptide further comprises at least 9 sequences which areinterspersed among the at least 10 sequences, wherein the at least 9sequences convey LCST transition behavior when the at least 10 sequencesconvey UCST transition behavior, and UCST transition behavior when theat least 10 sequences convey LCST transition behavior, such that thepolypeptide displays both LCST and UCST transition behavior.
 28. Thepolypeptide of claim 1, wherein the polypeptide exhibits a reversiblephase separation in response to a first stimulus and an irreversiblephase separation in response to a second different stimulus.
 29. Aenvironmentally responsive polypeptide comprising at least ten PGmotifs, and at least nine spacer sequences between the PG motifs, the atleast nine spacer sequences being between five and thirty amino acidresidues in length and not comprising a PG motif, and wherein thepolypeptide upon stimulation undergoes a conformational change that isaccompanied by aggregation.
 30. A method of effecting a conformationalchange in a polypeptide comprising exposing the polypeptide of claim 1to a stimulus such that the polypeptide undergoes a conformationalchange that is accompanied by aggregation or solubilization in responseto the stimulus.
 31. The method of claim 30, wherein the polypeptidebecomes bioactive or loses bioactivity following the conformationalchange.
 32. A method of effecting a conformational change in apolypeptide comprising exposing the polypeptide of claim 17 to astimulus such that the polypeptide undergoes a conformational changethat is accompanied by aggregation or solubilization in response to thestimulus.
 33. The method of claim 32, wherein the polypeptide becomesbioactive or loses bioactivity following the conformational change. 34.A method of effecting a conformational change in a polypeptidecomprising exposing the polypeptide of claim 21 to a stimulus such thatthe polypeptide undergoes a conformational change that is accompanied byaggregation or solubilization in response to the stimulus.
 35. Themethod of claim 34, wherein the polypeptide becomes bioactive or losesbioactivity following the conformational change.
 36. A nanoparticlecomprising the polypeptide of claim
 1. 37. The nanoparticle of claim 36,further comprising a therapeutic.
 38. The nanoparticle of claim 37,wherein the therapeutic is hydrophilic and is encapsulated by thenanoparticle.
 39. A method of delivering a therapeutic to a biologicalsite comprising contacting the biological site with the nanoparticle ofclaim 38, wherein the nanoparticle receives a stimulus at the biologicalsite and disassembles.
 40. The method of claim 39, wherein thenanoparticle comprises a proline-rich peptide which contacts thebiological site.
 41. The method of claim 39, wherein the biological sitecomprises an organ, tissue, tumor wound, disease or combination thereof.42. The method of claim 41, wherein the stimulus comprises an alterationin temperature, or altered pH.
 43. The nanoparticle of claim 37, whereinthe therapeutic is hydrophobic and the nanoparticle forms byself-assembling around the therapeutic.